Alina Vrieling
email: a.vrieling@dkfz-heidelberg.de

Katharina Buck

Rudolf Kaaks

Jenny Chang-Claude

Dr Ir Alina Vrieling

Adult Weight Gain in Relation to Breast Cancer Risk by Estrogen and Progesterone Receptor Status: a Meta-Analysis

Keywords: adult weight gain, breast cancer, estrogen receptor, progesterone receptor, meta-analysis

Purpose: Adult weight gain is positively associated with postmenopausal breast cancer and inversely associated with premenopausal breast cancer risk. To date, no meta-analysis has been conducted to assess this association by estrogen receptor (ER) and progesterone receptor (PR) status.

Methods: We searched PubMed for relevant studies published through March 2010.

Summarized risk estimates (RE) with 95% confidence intervals (CIs) were calculated using random effects or fixed effects models.

Results:

We retrieved nine articles on weight gain from adulthood to reference age and ER and/or PR defined breast cancer risk, reporting on three prospective cohort studies and eight case-control studies. Comparing the highest versus the lowest categories of adult weight gain, risk was increased for ER + PR + and ER + tumors combined (11 studies; RE = 2.03, 95% CI = 1.62, 2.45). Statistically significant heterogeneity (p heterogeneity = 0.002) was shown between risk estimates for a mixed population of pre-and postmenopausal women combined (4 studies; RE = 1.54, 95% CI = 0.86, 2.22) and for postmenopausal women only (7 studies; RE = 2.33; 95% CI = 2.05, 2.60). Risk for ER -PR -tumors among postmenopausal women was also slightly increased (7 studies, RE = 1.34, 95% CI = 1.06, 1.63), but statistically significantly different from risk for ER + PR + tumors (p heterogeneity 0.0001). No associations were observed for ER + PR -tumors whereas risk for ER -PR + tumors could not be assessed.

Conclusion:

The association between adult weight gain and postmenopausal breast cancer risk is heterogeneous according to ER/PR status and stronger for ER + PR + than for ER -PR -tumors.

INTRODUCTION

Body weight is an established risk factor for postmenopausal breast cancer whereas it is inversely associated with premenopausal breast cancer risk [START_REF]Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective[END_REF]. This may be due to differential effects of obesity on circulating endogenous estrogen levels according to menopausal status [START_REF] Potischman | Reversal of relation between body mass and endogenous estrogen concentrations with menopausal status[END_REF]. In postmenopausal women, estrogen is mainly synthesized by adipose tissue. Fatter women have larger adipose stores than leaner women, resulting in higher circulating levels of estrogen and lower levels of sex hormone-binding globulin (SHBG). In premenopausal women, estrogens mainly originate from ovarian synthesis, and levels are much higher than after menopause. However, overweight or obese premenopausal women are more likely to have anovulatory cycles and a greater clearance rate of estrogens by the liver compared to normal-weight women, resulting in lower levels of circulating estrogen and progesterone.

A recent meta-analysis has shown that the association between body mass index (BMI) and breast cancer risk is also heterogeneous according to estrogen receptor (ER) and progesterone receptor (PR) status [START_REF] Suzuki | Body weight and incidence of breast cancer defined by estrogen and progesterone receptor status--a meta-analysis[END_REF]. Higher BMI was associated with higher risk for ER + PR + tumors in postmenopausal women but had a protective effect on ER + PR + tumors in premenopausal women. By contrast, no associations were observed for ER -PR -tumors. Adult weight gain is thought to be a better measure to assess adiposity and its metabolic consequences than BMI [START_REF] Ballard-Barbash | Association of change in body mass with breast cancer[END_REF]. BMI reflects both lean and fat mass whereas adult weight gain largely reflects an increase in body fat independent of BMI [START_REF] Ballard-Barbash | Association of change in body mass with breast cancer[END_REF].

Several studies examining both adult weight gain and adult BMI in the same study population have found weight gain to be an equivalent [START_REF] Magnusson | Body size in different periods of life and breast cancer risk in post-menopausal women[END_REF][START_REF] Trentham-Dietz | Weight change and risk of postmenopausal breast cancer (United States)[END_REF] or stronger [START_REF] Ballard-Barbash | Association of change in body mass with breast cancer[END_REF][START_REF] Marchand | Body size at different periods of life and breast cancer risk[END_REF][START_REF] Brinton | Height and weight at various ages and risk of breast cancer[END_REF][START_REF] Huang | Dual effects of weight and weight gain on breast cancer risk[END_REF][START_REF] Feigelson | Weight gain, body mass index, hormone replacement therapy, and postmenopausal breast cancer in a large prospective study[END_REF] predictor of postmenopausal breast cancer risk than recent BMI. Adult weight gain has been inversely associated with premenopausal breast cancer risk [START_REF]Weight control and physical activity[END_REF].

Ten studies have investigated the association between adult weight gain and breast cancer risk according to ER/PR status of the tumor [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF][START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF][START_REF] Wenten | Associations of weight, weight change, and body mass with breast cancer risk in Hispanic and non-Hispanic white women[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF][START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF][19][20][START_REF] Colditz | Risk factors for breast cancer according to estrogen and progesterone receptor status[END_REF], and no systematic review or meta-analysis has been conducted thus far. Results of these studies were inconsistent, however, a limitation was that the number of non-ER + PR + tumors in individual studies was mostly small and comparisons for these groups were likely to be underpowered. Therefore, the aim of this meta-analysis was to investigate whether the association between adult weight gain and breast cancer risk differed according to ER/PR status of the tumor.

MATERIALS AND METHODS

Selection of studies

A systematic PUBMED search was performed to identify epidemiologic studies published through March 2010 that reported on the association between adult weight gain and risk for breast cancer defined by ER/PR status. We used a combination of the Medical Subject Headings (i.e., breast neoplasms, body weight, body mass index, estrogene receptors, progesterone receptors ) and also searched for related key words in title and abstract. The query retrieved 899 articles. In addition, cited references in retrieved articles were reviewed to identify possible further articles that may have been missed in this search. Eligible publications were assessed independently by two reviewers (AV, KB). Studies were included in the meta-analysis if they presented data from a prospective cohort or case-control study on the association between adult weight gain and ER and/or PR defined breast cancer risk. Studies that did not provide risk estimates (REs) for quantiles of adult weight gain were excluded.

Data extraction and classification

From each publication, we extracted the following information: the name of the first author, the year of publication, the country where the study was conducted, the major ER/PR assay, the name of the cohort study / the type of case-control study, years of follow-up (cohort studies), years of data collection (case-control studies), the number of overall cases, the study size (cohort studies), the number of controls (case-control studies), age, menopausal status, the weight gain categories studied, the effect estimates, and the adjusted factors. Relative risks or odds ratios and 95% confidence intervals (CI) from regression models were extracted and used for the subsequent analyses. The most fully adjusted risk estimates (REs) and confidence intervals for the highest quantile compared to the lowest quantile from each study were used for the meta-analyses. From the REs, the standard error of the estimate (SEE) was directly derived as SEE = [log (95% CI, upper limit)log (95% CI, lower limit)] / 3.92 and was employed in the calculation of the pooled estimates.

Statistical analysis

All analyses were carried out using the "meta" and "rmeta" packages of the statistical software environment R (version 2.7.1) [START_REF]R: A Language and Environment for Statistical Computing[END_REF]. The statistical analyses included a test of heterogeneity to determine whether the study results (of groups) were significantly heterogeneous. The correct model for the combination of single study results to a common pooled estimate could thereby be determined. Several statistical and quantifying measurements were used to assess heterogeneity of a set of point estimates together with the respective standard errors, i.e., the τ 2 heterogeneity estimator, the Q statistic yielding a (two-sided) p-value for heterogeneity, and the I 2 index [23].

Weighting was performed by the inverse variance method [START_REF] Hedges | Statistical methods for meta-analysis[END_REF] based on two commonly used models: the fixed effects model [START_REF] Whitehead | A general parametric approach to the meta-analysis of randomized clinical trials[END_REF] and the random effects model [START_REF] Dersimonian | Meta-analysis in clinical trials[END_REF]. In the prior model, one assumes that the effects are all in the same direction and of the same magnitude and are thus results of a common pooled effect. In the latter model, by contrast, there is no assumption that the effects are of identical direction and magnitude. Thus, in the random effects model the study results are combined in a more independent way by allowing variability within and heterogeneity between studies, respectively. Fixed effects models were used when heterogeneity was low (p-value > 0.1). Otherwise, random effects models were used. The individual study results as well as the combined pooled estimates were illustrated in forest plots. We calculated a pooled estimate for prospective cohort studies, casecontrol studies, and all studies combined. We initially included studies defined by joint ER/PR status and ER status only, and studies in mixed populations (pre-and postmenopausal women combined) and in postmenopausal women. Subsequently, we performed sensitivity analyses including only studies defined by joint ER/PR status or studies restricted to mixed populations or postmenopausal women, separately. For subgroup analyses, differences between the pooled REs were determined by the Q statistic.

In addition, a sensitivity analysis was conducted to investigate publication bias of the included studies. This was visualized using a funnel plot [START_REF] Light | Summing up. The science of reviewing research[END_REF][START_REF] Sterne | Funnel plots for detecting bias in meta-analysis: guidelines on choice of axis[END_REF] and quantified using weighted linear regression of the effect estimate on its SEE [START_REF] Egger | Bias in metaanalysis detected by a simple, graphical test[END_REF].

RESULTS

Characteristics of studies

We retrieved 10 articles on adult weight gain and risk of breast cancer by ER/PR status [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF][START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF][START_REF] Wenten | Associations of weight, weight change, and body mass with breast cancer risk in Hispanic and non-Hispanic white women[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF][START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF][19][20][START_REF] Colditz | Risk factors for breast cancer according to estrogen and progesterone receptor status[END_REF]. One article did not provide risk estimates for quantiles of adult weight gain, and was therefore excluded [START_REF] Colditz | Risk factors for breast cancer according to estrogen and progesterone receptor status[END_REF]. In total, 9 articles (including 11 studies) on adult weight gain (10 from early adulthood, 1 from age 50 to reference age) and risk of breast cancer by ER/PR status were included in the meta-analysis (Table 1). Three of these studies were prospective cohort studies [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF], and eight were population-based case-control studies [START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF][START_REF] Wenten | Associations of weight, weight change, and body mass with breast cancer risk in Hispanic and non-Hispanic white women[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF][START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF][19][20]. Three case-control studies defined breast tumours by ER status only [START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF]20], and nine studies by joint ER/PR status [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF][START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF][START_REF] Wenten | Associations of weight, weight change, and body mass with breast cancer risk in Hispanic and non-Hispanic white women[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF]19]. Four studies were conducted in a mixed population of pre-and postmenopausal women [START_REF] Wenten | Associations of weight, weight change, and body mass with breast cancer risk in Hispanic and non-Hispanic white women[END_REF]20], there were no studies that included only premenopausal women, and seven studies included only postmenopausal women [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF][START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF][START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF][19]. All studies were conducted in the US, except for one study that was conducted in Sweden [19]. The number of cases with known ER/PR status ranged from 170 to 651 among the prospective cohort studies, and from 210 (ER + PR + and ER -PR -only) to 1,001 among the case-control studies.

ER + PR + tumors

Random effects models were used to assess the association between adult weight gain and ER-positive breast cancer risk. Adult weight gain was associated with an increased risk for ER + PR + and ER + tumors combined [START_REF]Weight control and physical activity[END_REF]

ER -PR -tumors

Fixed effects models were used to assess the association between adult weight gain and ER-negative breast cancer risk. Adult weight gain was associated with an increased risk for ER -PR -and ER -tumors combined (11 studies; RE = 1.36, 95% CI = 1.14, 1.58, p heterogeneity = 0.14) (Figure 2, Table 2).

Results were not statistically significantly heterogeneous (p heterogeneity = 0.92)

for the mixed population of pre-and postmenopausal women combined (4 studies; RE = 1.32, 95% CI = 0.74, 1.89, p heterogeneity = 0.06) and for postmenopausal women only (7 studies; RE = 1.34; 95% CI = 1.06, 1.63, p heterogeneity = 0.30).

ER + PR + versus ER -PR -tumors

Although risk among postmenopausal women was statistically significantly increased for both ER + PR + and ER -PR -tumors, adult weight gain was associated with a statistically significantly greater risk for ER + PR + than ER -PR -tumors (p heterogeneity 0.0001).

ER + PR -and ER -PR + tumors

The association between adult weight gain and risk for ER + PR -breast tumors was only assessed in postmenopausal women. Fixed effects models were used and no statistically significant association was found (4 studies; RE = 1.04, 95% CI = 0.68, 1.40, p heterogeneity = 0.81). Associations for ER -PR + tumors could not be assessed due to the small number of studies and the limited number of cases (3 studies).

Publication bias

Funnel plots were relatively symmetrical (data not shown) and no evidence for publication bias was found for ER + PR + , ER -PR -and ER + PR -tumors (Egger's test: p = 0.96, p = 0.19, and p = 0.30, respectively).

DISCUSSION

This meta-analysis shows that the association between adult weight gain and postmenopausal breast cancer risk is heterogeneous according to ER/PR status of the tumor. Higher adult weight gain was associated with higher risk for ER + PR + postmenopausal breast cancer and risk for ER -PR -postmenopausal breast cancer was only slightly increased. No association with ER + PR -postmenopausal breast cancer risk was observed.

A previous meta-analysis on BMI found that higher BMI was associated with higher risk for ER + PR + tumors in postmenopausal women, but no t with risk for ER - PR -tumors [START_REF] Suzuki | Body weight and incidence of breast cancer defined by estrogen and progesterone receptor status--a meta-analysis[END_REF]. In our current meta-analysis, adult weight gain was associated with higher risk for both ER + PR + and ER -PR -postmenopausal breast cancer, although risk was statistically significantly higher for ER + PR + compared to ER -PR -tumors.

Both BMI [START_REF] Suzuki | Body weight and incidence of breast cancer defined by estrogen and progesterone receptor status--a meta-analysis[END_REF] and adult weight gain were not associated with ER + PR - postmenopausal breast cancer risk. The association of BMI [START_REF] Suzuki | Body weight and incidence of breast cancer defined by estrogen and progesterone receptor status--a meta-analysis[END_REF] and adult weight gain with ER -PR + postmenopausal breast cancer could not be reliably assessed due to the limited number of studies. No studies were available on adult weight gain and risk of premenopausal breast cancer by ER/PR status, and therefore this association could not be independently assessed.

BMI is the most commonly used anthropometric measure to define obesity, and has been consistently associated with increased postmenopausal breast cancer risk [START_REF]Food, Nutrition, Physical Activity, and the Prevention of Cancer: a Global Perspective[END_REF]. However, adult weight gain is thought to be a be tter measure to assess adiposity and its metabolic consequences than BMI. Whereas BMI reflects both lean body mass and adipose mass, weight gain in adult life is generally a gain of body fat and thus potentially represents the age-related metabolic change that may be important in breast cancer development [START_REF] Ballard-Barbash | Association of change in body mass with breast cancer[END_REF]. Several studies examining both adult weight gain and adult BMI in the same study population have found weight gain to be an equivalent [START_REF] Magnusson | Body size in different periods of life and breast cancer risk in post-menopausal women[END_REF][START_REF] Trentham-Dietz | Weight change and risk of postmenopausal breast cancer (United States)[END_REF] or stronger [START_REF] Ballard-Barbash | Association of change in body mass with breast cancer[END_REF][START_REF] Marchand | Body size at different periods of life and breast cancer risk[END_REF][START_REF] Brinton | Height and weight at various ages and risk of breast cancer[END_REF][START_REF] Huang | Dual effects of weight and weight gain on breast cancer risk[END_REF][START_REF] Feigelson | Weight gain, body mass index, hormone replacement therapy, and postmenopausal breast cancer in a large prospective study[END_REF] predictor of postmenopausal breast cancer risk than recent BMI.

In postmenopausal women, obesity has been associated with hyperinsulinaemia and insulin resistance, and with elevated estradiol and lower SHBG levels [START_REF] Calle | Overweight, obesity and cancer: epidemiological evidence and proposed mechanisms[END_REF]. Similar associations have been found for adult weight gain [START_REF] Stoll | Timing of weight gain in relation to breast cancer risk[END_REF].

Hyperinsulinaemia and insulin resistance, among other factors, may promote both ER + PR + and ER -PR -breast tumors in postmenopausal women. The stronger associations found for adult weight gain in relation to risk for ER + PR + compared with ER -PR -postmenopausal breast cancer may be explained by an additional promoting effect of higher free estradiol levels on ER + PR + breast tumors, specifically.

Although this meta-analysis shows that adult weight gain is clearly associated with increased risk for ER + PR + breast cancer and to a lesser extent with increased risk for ER -PR -breast cancer in postmenopausal women, we observed large heterogeneity between the study-specific risk estimates, in particular for ER + PR + breast tumors. One possible explanation for this heterogeneity may be differences in the definition of quantiles between studies, which preclude relating risk for ER + PR + or ER -PR -breast tumors to specific cut-off measurements of adult weight change.

However, the heterogeneity between studies may be due to several ot her reasons.

Studies were performed in different age groups and adult weight gain has been previously shown to be differentially related to premenopausal and postmenopausal breast cancer risk [START_REF]Weight control and physical activity[END_REF]. Indeed, restricting the studies to those conducted in postmenopausal women reduced heterogeneity.

Further, there is a potential for measurement error from self-reported weight and recall of weight during early adulthood. However, correlations between measured and self-reported weight typically are 0.95 [START_REF]Weight control and physical activity[END_REF], and correlations between recalled weight over several decades and measured weight have been between 0.80 and 0.87 in several studies [START_REF] Willett | Nutritional epidemiology[END_REF].

The assay methods for the measurement of ER/PR status and cutoff points for ER/PR status were not available for all studies and also varied across/within studies. This may have led to misclassification of the tumor subtypes and in consequence to attenuation of the observed risk estimates. Some studies only reported on ER and PR status separately whereas other studies considered joint ER/PR status. The combination of ER and PR status is reflective of true tumor estrogen dependence, since tumors will be both ER and PR positive if expression of the PR gene is induced by the presence of a normally functioning ER-estradiolbinding system [START_REF] Rose | Influence of obesity on breast cancer receptor status and prognosis[END_REF]. Restricting our meta-analyses to studies measuring joint ER/PR status increased the risk estimates.

Current use of hormone replacement therapy increases circulating estrogen levels and may obscure the effect of adult weight gain on postme nopausal breast cancer risk. Some studies restricted their study population of postmenopausal women to non-users of postmenopausal hormones [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF]19] while other studies adjusted for postmenopausal hormone use [START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF][START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF]. However, results for adult weight gain and postmenopausal breast cancer risk did not appear to differ between the two types of studies.

Associations may be different for different ethnic groups. In black women, adult weight gain was not statistically significantly associated with ER + PR + postmenopausal breast cancer risk [START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF]. It may also be possible that it was more difficult to detect this association in black women due to the smaller study population and the lower proportion of breast carcinomas that are ER + , PR + , or both.

To account for baseline differences in body size, some studies adjusted the analyses for baseline weight and height or BMI [START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Palmer | A prospective study of body size and breast cancer in black women[END_REF][START_REF] Wenten | Associations of weight, weight change, and body mass with breast cancer risk in Hispanic and non-Hispanic white women[END_REF][START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF]. The other studies either adjusted for recent BMI [START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF]19], or did not perform any adjustment for body size [START_REF] Feigelson | Adult weight gain and histopathologic characteristics of breast cancer among postmenopausal women[END_REF][START_REF] Enger | Body size, physical activity, and breast cancer hormone receptor status: results from two case-control studies[END_REF]20]. However, results did not appear differ for studies with or without adjustment. Finally, unknown or residual confounding may be present.

The relation between adult weight gain and ER/PR defined breast cancer risk

has not yet been fully clarified. To the best of our knowledge, no studies have been conducted in premenopausal women only. Future studies should investigate whether adult weight gain has a differential effect on ER + PR + premenopausal breast cancer risk, as is suggested by the lower risk estimates in the mixed population of pre-and postmenopausal women combined compared to those in postmenopausal women.

Further, it is unclear whether adult weight gain throughout the entire adulthood lifespan or weight gain during specific life stages is most relevant for the observed increase in ER + PR + defined postmenopausal breast cancer risk. Weight gain during pubertal years is distributed primarily on the hips and buttocks . During later adult life fat accumulates preferentially around the waist. Only few studies ha ve addressed the effect of adult weight gain during multiple life stages with respect to overall postmenopausal breast cancer risk. Three prospective cohort studies and one case-control study showed that both adult weight gain before and after menopause were associated with postmenopausal breast cancer risk [START_REF] Ahn | Adiposity, adult weight change, and postmenopausal breast cancer risk[END_REF][START_REF] Han | Lifetime adult weight gain, central adiposity, and the risk of pre -and postmenopausal breast cancer in the Western New York exposures and breast cancer study[END_REF][START_REF] Harvie | Association of gain and loss of weight before and after menopause with risk of postmenopausal breast cancer in the Iowa women's health study[END_REF][START_REF] Eliassen | Adult weight change and risk of postmenopausal breast cancer[END_REF] whereas in one case-control study an increase in risk was only evident for adult weight gain after menopause [START_REF] Eng | Body size changes in relation to postmenopausal breast cancer among women on Long Island, New York[END_REF]. None of these studies investigated whether these associations differed by ER/PR status of the tumor.

In conclusion, the association between adult weight gain and postmenopausal breast cancer risk is heterogeneous according to ER/PR status of the tumor, with higher risk estimates for ER + PR + than for ER -PR -tumors. The association of adult weight gain with ER/PR defined premenopausal breast cancer risk could not be independently assessed. These findings suggest that control of body weight may prevent ER + PR + tumors, and to a lesser extent ER -PR -tumors, after menopause.

  b p-values (two-sided) were based on the Q statistic of heterogeneity. c I 2 quantifies the percentage of variation across studies that is due to heterogeneity rather than chance. FEM = fixed effects model, mixed = pre -and postmenopausal women combined, REM = random effects model.

Figure 1 Figure 2

 12 Figure 1 Association between adult weight gain and ER + PR + breast cancer risk

Table 1

 1 Characteristics of prospective cohort and case-control studies on weight gain from age 15, 18, 20 or 50 to referent age and breast cancer incidence by ER/PR status Age, education, age at menarche, age at first birth, previous benign breast di sease, family history of breast cancer, age at menopause,

	Author, year,	Design (study	Age and	Type of	ER/PR defined	Exposure measurement (kg	Effect	Adjusted factors
	country, major	name), follow-up /	menopau-	tumor	cases number	unless otherw ise stated)	estim ates
	ERPR assay	yr-data collection,	sal status		(approxim ate %
	[ref]	no. of overall			known receptor)
		cases and (study
		size) / controls
	Prospective cohort studies
	Feigelson,	Prospective coh	50-74 yr	ER+PR+	445 cases (68%)		27.3 vs. -2.3 to 9.1 from age 18	2.42 (1.82, 3.23)	Age, age at menarche, age at menopause,
	2006, US, N.A. [12]	(CPS-II), 1992-2001,	Post	ER+PR-/ ER-PR+	108 cases (17%)		27.3 vs. -2.3 to 9.1 from age 18	1.32 (0.70, 2.49)	number of live births, age at first live birth, use of OC, family history of breast cancer,
		1,200 cases (44,161)		ER-PR-	98 cases (15%)		27.3 vs. -2.3 to 9.1 from age 18	1.78 (0.98, 3.23)	history of breast cysts, history of mammography, height, education, physical
									activity, alcohol intake, aspirin use, race
									(excluded PMH user).
	Ahn, 2007, US,	Prospective coh	50-71 yr	ER+PR+	202 cases (66%)		30 vs. -2.0 to 9.9 from age 18	2.69 (1.74, 4.17)	Age, age at menarche, age at menopause,
	N.A. [13]	(AARP), 1996-2000, 2,111 cases (99,039)	Post	ER+PR-ER-PR+ ER-PR-	44 cases (14%) 10 cases (3%) 53 cases (17%)		30 vs. -2.0 to 9.9 from age 18 30 vs. -2.0 to 9.9 from age 18 30 vs. -2.0 to 9.9 from age 18	1.28 (0.47, 3.48) -0.61 (0.21, 1.82)	age at first live birth, parity, smoking, education, race, family history of breast cancer, fat intake, alcohol intake, oophorectomy, physical activity, weight at
									age 18, height (excluded PMH user).
	Palmer, 2007,	Prospective coh	25-79 yr	ER+PR+	82 cases (48%)		25 vs. 15 from age 18	1.29 (0.73, 2.28)	Age, age at menarche, parity, age at fi rst
	US, N.A. [14]	(BWHS),	Post	ER+PR-/	36 cases (21%)		25 vs. 15 from age 18	0.31 (0.13, 0.77)	birth, age at menopause, vigorous acti vity,
		1995-2005,		ER-PR+					education, family history of breast cancer,
		442 cases (9,542)		ER-PR-	52 cases (31%)		25 vs. 15 from age 18	1.03 (0.52, 2.05)	BMI at age 18 (excluded PMH user).
	Case-control studies
	Enger, 2000,	Population-based	55-64 yr	ER+PR+	450 cases (59%)		29.2 vs. 0 % from age 18	2.32 (1.58, 3.41)	Age at reference year (matched),
	US, DCC [15]	cc 1987-1989	Post	ER+PR-ER-PR+	159 cases (21%) 24 cases (3%)	-	29.2 vs. 0 % from age 18	0.99 (0.58, 1.78) -	socioeconomic status (matched), number of full term pregnancy, breast feeding, age at
		760 cases 1,091 controls		ER-PR-	127 cases (17%)		29.2 vs. 0 % from age 18	1.75 (0.91, 3.38)	menopause, use of PMH, family history of breast cancer, al cohol intake, physical
									activity.
	Wenten, 2002,	Population-based	30-74yr						Age, family history of breast cancer, total
	US, N.A. [16]	cc	Mixed						METS, parity, use of OC, breast feeding, age
		1992-1994	NHW	ER+PR+	133 cases		14 vs. 4 from age 18	1.05 (0.60, 1.84)	at first birth, use of PMH, menopausal status,
		694 cases		ER-PR-	77 cases		14 vs. 4 from age 18	0.93 (0.42, 2.05)	weight at age 18.
		813 controls	Hispanic	ER+PR+ ER-PR-	133 cases 77 cases		14 vs. 4 from age 18 14 vs. 4 from age 18	3.04 (1.47, 6.26) 1.73 (0.71, 4.25)
	Eng, 2005, US,	Population-based	20-98 yr	ER+PR+	387 cases (58%)		11.3 vs. 0 from age 50	2.17 (1.38, 3.42)	Age at reference date, number of pregnancy,
	N.A. [17]	cc	Post	ER+PR-	121 cases (18%)		11.3 vs. 0 from age 50	1.25 (0.57, 2.74)	use of PMH, family history of breast cancer,
		1996-1997 990 cases 1,006 controls		ER-PR+ ER-PR-	30 cases (4%) 132 cases (20%)		11.3 vs. 0 from age 50 11.3 vs. 0 from age 50	1.54 (0.38, 6.35) 1.50 (0.72, 3.13)	history of benign breast disease, BMI at age 50 yr.
	Han, 2006, US,	Population-based	35-79 yr	ER+	510 cases (79%)		27.3 vs. 0 to 9.1 from age 20	2.42 (1.62, 3.61)
	DCC/IHC [18]	cc 1996-2001	Post	ER-PR+	136 cases (21%) 389 cases (60%)		27.3 vs. 0 to 9.1 from age 20	1.19 (0.58, 2.43) 3.14 (1.96, 5.04)

Table 2

 2 Results of the meta-analysis for adult weight gain and ER + PR + and ER -PR -

	breast cancer risk
	Group	No. of	τ 2 a p heterogeneity	b	I 2 c	Model	Pooled estimate
		studies					used	(95% CI)
	Adult weight change, ER+PR+
	All studies	11	0.44	0.0001		91%	REM	2.03 (1.62, 2.45)
	Mixed	4	0.42	0.0001		88%	REM	1.54 (0.86, 2.22)
	Postmenopausal	7	0.10	0.0024		71%	REM	2.33 (2.05, 2.60)
	Adult weight change, ER-PR-
	All studies	11	0.07	0.14		32%	FEM	1.36 (1.14, 1.58)
	Mixed	4	0.20	0.06		60%		1.32 (0.74, 1.89)
	Postmenopausal	7	0.03	0.30		17%	FEM	1.34 (1.06, 1.63)

a τ 2 is an estimator of between-studies variance.

ACKNOWLEDGEMENTS

This work was funded by the Deutsche Krebshilfe, project number 108253. The authors had no conflict of interest.


Ruben Ghillebert

Erwin Swinnen

Pepijn De Snijder

Bart Smets

Joris Winderickx
email: joris.winderickx@bio.kuleuven.

Differential roles for the low-affinity phosphate transporters Pho87 and Pho90 in Saccharomyces cerevisiae

Keywords: Saccharomyces cerevisiae, nutrient signaling, Pho87, Pho90, vacuolar targeting, non-redundancy

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Introduction

As inorganic phosphate (Pi) is an essential nutrient for all organisms, maintaining proper Pi homeostasis is critical to ensure survival of cells and organisms. A crucial step in controlling Pi homeostasis encompasses the transport of Pi across the plasma membrane into the cell. Since cellular membranes are impermeable to Pi, specialised integral membrane proteins are required to transport this essential compound. In Saccharomyces cerevisiae, two Pi-symport systems exist [START_REF] Borst-Pauwels | Ion transport in yeast[END_REF][START_REF] Persson | Phosphate permeases of Saccharomyces cerevisiae: structure, function and regulation[END_REF][START_REF] Tamai | Regulation of inorganic phosphate transport systems in Saccharomyces cerevisiae[END_REF]. The low-affinity transport system has a K m for external Pi of approximately 1 mM and satisfies the cellular need for Pi at normal or high Pi concentrations. This low-affinity Pi-transport system is believed to be composed of the partially redundant Pho87 and Pho90 H + /Pi-symporters [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF]. A third gene, PHO91, was initially considered to be a third component of the low-affinity plasma membrane phosphate transport system [START_REF] Wykoff | Phosphate transport and sensing in Saccharomyces cerevisiae[END_REF], but later studies localized the Pho91 protein at the vacuolar membrane, indicating that this protein is not directly involved in the acquisition of phosphate from the external medium [START_REF] Hurlimann | Pho91 Is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae[END_REF]. The high-affinity Pi-transport system has a K m of about 10 µM and is sustained by the Pho84 and Pho89 high-affinity H + /Pi-and Na + /Pi-symporters [START_REF] Bun-Ya | The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter[END_REF][START_REF] Martinez | Physiological regulation of the derepressible phosphate transporter in Saccharomyces cerevisiae[END_REF]. While expression of the PHO87, PHO90 and PHO91 genes was shown to be independent of Pi concentrations, transcription of PHO84 and PHO89 is induced when Pi becomes limiting, a process controlled by the PHOpathway [START_REF] Auesukaree | Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae[END_REF]. Signal flow through this PHO-pathway is regulated by the cyclin-dependent kinase inhibitor (CKI) Pho81. Under Pi-starving conditions, activation of this CKI leads to inhibition of the Pho85-Pho80 cyclin-dependent kinase complex and nuclear translocation of the dephosphorylated transcription factor Pho4. Nuclear Pho4 then induces a set of genes known as the PHO-genes [10], which includes PHO84 and PHO89. When sufficient amounts of extracellular Pi are available, Pho81 is inactivated and this results in an active Pho85-Pho80 kinase complex that is able to phosphorylate and trigger the nuclear exclusion of Pho4, with as consequence the repression of the PHO-genes [START_REF] Wanke | Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex[END_REF]. An additional role for the Pho85-Pho80 complex is to regulate the localization and hence the activation of the Rim15 protein kinase in response to fluctuating Pi levels [START_REF] Wanke | Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex[END_REF]. Rim15 is essential for proper entry into stationary phase as it controls the activity of the transcription factors Msn2,4 and Gis1, which are required for expression of the so-called Stress-Responsive-Element (STRE) genes and the Post-Diauxic-Shift (PDS) genes, respectively [12][START_REF] Cameroni | The novel yeast PAS kinase Rim 15 orchestrates G0-associated antioxidant defense mechanisms[END_REF][START_REF] Roosen | PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability[END_REF]. Rim15 acts as a nutritional integrator in yeast, since it is regulated by at least four nutrient-regulated kinases: PKA inhibits Rim15 activity [START_REF] Reinders | Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase[END_REF], while TORC1 and Sch9 promote cytoplasmic retention [16,[START_REF] Wanke | Caffeine extends yeast lifespan by targeting TORC1[END_REF] and the Pho85-Pho80 complex promotes its nuclear exclusion [START_REF] Wanke | Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex[END_REF][START_REF] Swinnen | Rim15 and the crossroads of nutrient signalling pathways in Saccharomyces cerevisiae[END_REF]. Under Pi-starving conditions, inhibition of the Pho85-Pho80 kinase by Pho81 results in an active, nuclear Rim15 and proper entry into stationary phase. In contrast, under high Pi conditions, phosphorylation of Rim15 by the Pho85-Pho80 kinase induces its nuclear exclusion thereby leading to its inactivation. In line with this, we already showed that the Pho85-Pho80 complex mediates Pi-dependent activation of typical PKA-targets like activation of trehalase, mobilisation of trehalose and repression of stress-responsive genes and that the regulation of many of these properties occurs via a Pho85-Pho80-dependent inhibition of Rim15 activity [19].

The ability of inorganic Pi to act as a nutrient signal affecting PKA targets is closely correlated to its uptake into the cell, as inactivation of Pi-transport by destruction of the proton gradient across the plasma membrane with the protonophore 2,4-dinitrophenol completely prevented Pi-induced trehalase activation [20]. Although the predominant Pitransport activity present in Pi-starved cells is represented by the Pho84 transporter [START_REF] Bun-Ya | The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter[END_REF][START_REF] Bun-Ya | Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae[END_REF], which is additionally exemplified by the inability of a pho84Δ strain to mediate rapid Pisignaling [START_REF] Lundh | Molecular mechanisms controlling phosphateinduced downregulation of the yeast Pho84 phosphate transporter[END_REF][START_REF] Popova | Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor[END_REF], we previously observed clear activation of trehalase after addition of Pi to cells deleted for PHO4 or PHO81, in which no PHO84 transcription can be detected [19]. Additionally, both Pho84 and Pho87 have been shown to act as specific Pi sensors for rapid Pi-signaling [20], but the genetic and physiological relevance of Pi-signaling through Pho87 was never investigated. Our data reveals that in the absence of Pho4, the low affinity Pitransporter Pho87 becomes essential for Pi-signaling. We show that in Pi-starved cells, the low-affinity Pi-transporters Pho87 and Pho90 are normally subjected to vacuolar targeting, but in cells devoid of Pho4 activity, this process is specifically hampered for Pho87 due to the lack of expression of SPL2, which was previously identified as a negative regulator of lowaffinity Pi-transport and was shown to interact with the N-terminal SPX-domain of both Pho87 and Pho90 [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF][START_REF] Wykoff | Positive feedback regulates switching of phosphate transporters in S. cerevisiae[END_REF]. However, we now demonstrate that, in contrast to Pho87, the vacuolar targeting of the Pho90 upon Pi-starvation is independent of both Pho4 activity and Spl2. Furthermore, when cells are starved for other essential nutrients or are treated with rapamycin, the vacuolar targeting applies to both low-affinity Pi-transporters and also this process is independent of Pho4 and Spl2. Nonetheless, the vacuolar targeting of both Pho87 and Pho90 under these conditions still depends on their N-terminal SPX-domain. This indicates that besides Spl2, other, yet unidentified, proteins must interact with this domain in order to fine-tune the presence of the low-affinity Pi-transporters at the plasma membrane in response to a multitude of nutrient signals. 1. Deletions were made using polymerase chain reaction-based disruption cassettes as described previously [START_REF] Brachmann | Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications[END_REF]. Yeast was transformed with the Gietz method [START_REF] Gietz | Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure[END_REF]. Sporulation was performed by spotting diploid cells on plates containing 1% K-acetate, 0.1% KHCO 3 , pH 6.0 for 5 to 6 days at 24°C. Tetrads were dissected and separated in a raster using a micromanipulator (Singer Instruments). The P TEF -EGFP-PHO87, P TEF -EGFP-PHO90, P TEF -EGFP-PHO87375N and P TEF -EGFP-PHO90375N were kindly provided by Hurlimann et al. [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF][START_REF] Hurlimann | Pho91 Is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae[END_REF].

Culture and starvation conditions

Yeast cells were grown in synthetic complete minimal medium containing 0,5% w/v (NH 4 ) 2 SO 4 , 1,9g/l yeast nitrogen base w/o amino acids, supplemented with all amino acids and 2% w/v glucose (ScD). For monitoring growth on ScD agar with fluctuating extracellular Pi levels, a preculture on ScD was inoculated in fresh ScD medium at an initial OD 600 of 0.1 and tenfold dilutions were spotted on phosphate starvation medium (5.7 g/l of yeast nitrogen base with ammonium sulphate without phosphate) with 2% glucose, required amino acid/nucleotide supplements and with known Pi concentration. Pictures were taken after 2-3 days. For growth experiments with arsenate, cells were grown in standard rich medium with 2% w/v glucose (YPD) and spotted on YPD or on YPD with an additional 10mM Pi and with or without 5mM AsO4. For phosphate signaling and consumption experiments, yeast cells were grown into mid-exponential phase (OD 600 = 1.5-2) on ScD medium, harvested, washed two times and transferred to phosphate starvation medium with 4% glucose and required amino acid/nucleotide supplements. Cells were starved for phosphate for 3 days, during which starvation medium was refreshed daily. Before re-addition of phosphate, the phosphatestarved, glucose-repressed cells were rapidly cooled on ice and harvested by centrifugation. The pellet was washed twice with ice-cold Mes-buffer pH 6.0 and resuspended in fresh phosphate starvation medium with 4% glucose at a cell density of 25mg cells (wet weight) per ml. After 30 min of incubation, KH 2 PO 4 was added to the culture and samples were taken at the indicated time points. For monitoring growth recovery after re-addition of phosphate, the Pi-starved cells were resuspended in fresh Pi-starvation medium supplemented with KH 2 PO 4 at an initial OD 600 of 0.1 and growth was followed by measuring OD 600 at the indicated time points. For the localization studies, cells were grown till mid-exponential phase in ScD and then starved for phosphate and/or glucose or treated with 200nM rapamycin for 6-8 hours unless indicated otherwise. All incubations were done at 30°C under continuous shaking. Sampling, extraction and determination of trehalase activity Samples of 50 mg cells (wet weight) were taken at the indicated time-points. Cells were rapidly cooled by addition of ice-cold water, centrifuged and resuspended in 500 μl of 25 mM MES buffer, 50 μM CaCl 2, pH 7 for extraction. Crude cell extracts were prepared as described previously [20] and dialyzed (BRL microdialysis system) against 25 mM MES buffer pH 7 with 50 μM CaCl 2 at 4°C. Trehalase activity in dialyzed cell extracts was determined as described previously [20]. The specific activity is expressed as nmol glucose liberated per min and per mg protein. Total amount of protein in the samples was determined using a standard method [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF]. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Phosphate-consumption assay

After addition of 1 mM KH 2 PO 4 to Pi-starved cells, 1 ml samples were taken and cells were rapidly pelleted at 4°C. Phosphate levels in the supernatans were determined with the Fiske-Subbarow colorimetric method for the determination of inorganic phosphate [START_REF] King | The colorimetric determination of phosphorus[END_REF].

RNA extraction and Northern blot analysis

Samples of 120 mg cells (wet weight) were taken at the indicated time-points and rapidly cooled by addition of ice-cold water. The cells were pelleted and washed once with ice-cold water and stored at -80°C. Isolation of total RNA was performed as described previously [20].

Probes were labeled with α-32 P-dCTP using the High Prime kit (Boehringer Mannheim). Northern blots were made by separation of total RNA on gels containing 1% (w/v) agarose in 50 mM boric acid, 1 mM sodium citrate, 5 mM NaOH pH 7.5 and 1% formaldehyde. Subsequently, RNA was transferred by capillary blotting to a Hybond-N membrane (Amersham) using 10× SCC buffer (1.5 M NaCl, 0.15 M Na-citrate, pH 7.0). The filters were hybridized with the 32 P-labelled probes consisting of fragments of the desired coding region. The blots were analyzed using PhosphorImager technology (FUJIX, BAS-1000). Fluorescence microscopy Cells expressing EGFP-fusion constructs were grown as described above and used directly for microscopy without fixation. Cells were viewed using a 'Leica DM4000B' fluorescence microscope. Pictures were taken with a 'Leica DFC420C' camera using the Leica Application Suite software. For FM4-64 ([N-(3-triethylammoniumpropyl)-4-(p-diethylamino phenylhexatrienyl) pyridinium dibromide; Molecular Probes), which specifically stains the vacuolar membrane, strains overexpressing EGFP-fused Pho87 and Pho90 were starved for 6 hours for phosphate. The cells were stained with FM4-64 dye for 20 min at 4°C and incubated at RT for 60 minutes to induce transport of FM4-64 via endocytotic intermediates to the vacuolar membrane and fluorescence of both the FM4-64 and the EGFP-fusions was examined. Overlaying of images was performed with ImageJ.

Reproducibility of the results

All experiments were performed at least three times. For growth curves, trehalase and Piconsumption experiments, representative figures are shown. The absolute values are somewhat variable between different experiments, but the relative differences observed between separate experiments and strains are highly reproducible. For Northern blot experiments, representative blots from three independent experiments are shown.

Results

The deletion of PHO4 shifts rapid Pi-signaling control from Pho84 to Pho87

Recent papers have demonstrated that Pho84 is the major Pi-transporter in S. cerevisiae involved in rapid Pi-signaling [START_REF] Lundh | Molecular mechanisms controlling phosphateinduced downregulation of the yeast Pho84 phosphate transporter[END_REF][START_REF] Popova | Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor[END_REF], as the pho84Δ strain is unable to rapidly reverse Pistarvation responses. However, we previously observed rapid phosphate signaling in strains deleted for PHO4 or PHO81 [19], both of which do not display expression of PHO84. Thus, either expression of PHO84 below detection levels is sufficient to sustain phosphate signaling or another phosphate transporter takes over the function of Pho84 in the absence of Pho4 activity. To investigate this in more detail, the individual Pi-transporters were deleted in a pho4Δ background. Upon re-addition of KH 2 PO 4 to Pi-starved cultures, we observed that additional deletion of PHO4 not only restored Pi-signaling in pho84 cells, but it also shifted the Pi-signaling control towards Pho87. The latter is illustrated by the absence of trehalase activation in a pho4 pho87 strain (Fig. 1A), which nicely correlated with an inability to rapidly consume measurable amounts of Pi (Fig. 1B). Rapid Pi-signaling functions in part to quickly restart growth of the yeast cell, which is further highlighted by the rapid induction of ribosomal protein genes and the repression of the stress-responsive genes (Fig. S1A). Consequently, we observed that the pho84Δ and the pho4 pho87 mutant display retardation in growth resumption after addition of phosphate to Pi-starved cells when compared to the wild-type strain and the other mutants (Fig. S1B). This nicely correlates with the delayed induction of ribosomal protein genes and the delayed inhibition of stress-responsive genes in these strains (Fig. S1A). Notably, the presence of the other low-affinity Pi-transporter, i.e. Pho90, does not substitute for Pho87 when it comes to Pi-signaling in the pho4 strain (Fig. 1), indicating that the two low-affinity Pi-transporters are non-redundant. Deletion of PHO91 in a pho4Δ did also not affect rapid Pi-signaling under these conditions (results not shown). Pho91 was not further investigated as this low-affinity Pi-transporter was shown to mediate Pi-transport across the vacuolar membrane [START_REF] Hurlimann | Pho91 Is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae[END_REF]. Together, the above described results indicate that specifically the Pho87 Pi-transporter mediates Pi-signaling in the absence of a functional Pho4 transcription factor. One possibility would be that Pho4 is somehow involved in transcriptional repression of PHO87. However, previous studies already indicated that transcription of the low-affinity Pitransporters is independent of both Pi concentration and Pho4 activity [START_REF] Auesukaree | Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae[END_REF]. This was further confirmed in our study, since there was no significant alteration in the expression of either PHO87 or PHO90 when wild-type, pho84 or pho4 cells were starved for Pi, this in contrast to the expression of PHO84 and PHO89, encoding the high-affinity Pi-transport system (Fig. S1C). Altogether, these results imply that under Pi-limiting conditions, wild-type yeast cells downregulate the low-affinity Pi-transporter Pho87 and turn to the high-affinity Pho84 Pitransporter to signal and take up the available Pi. Both processes depend on the activation of the Pho4 transcription factor, which directly induces PHO84 expression and results in the inactivation of Pho87 at a posttranscriptional level. Spl2 mediates Pi-starvation-induced vacuolar targeting of Pho87, but not of Pho90. In yeast, several plasma membrane nutrient transporters are inactivated and targeted to the vacuole in response to changes in the availability of nutrients [START_REF] Springael | Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae[END_REF][START_REF] Magasanik | Nitrogen regulation in Saccharomyces cerevisiae[END_REF][31][START_REF] Daicho | The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast[END_REF]. To determine whether the low-affinity Pi-transporters Pho87 and Pho90 are subject to nutrient limitation-induced vacuolar targeting as well and to establish a role of Pho4 in this process, we set out to compare the intracellular localization of the EGFP-tagged Pho87 and Pho90 proteins under different nutritional conditions in the wild-type strain and in the pho4 mutant. For this, we opted to use the strains previously described [START_REF] Hurlimann | Pho91 Is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae[END_REF], where chromosomal N-terminal EGFPfusion genes of PHO87 and PHO90 were expressed by the TEF promoter. As shown in Fig. 2A, Pho87 was localized at the plasma membrane of wild-type cells grown on ScD medium, but this low-affinity transporter is endocytosed and targeted to the vacuole when cells encountered Pi-starvation. This was additionally confirmed by the deletion of END3, encoding an essential component for endocytosis, which prevented the internalisation of Pho87 and, in consequence, led to the stabilization of this Pi-transporter at the plasma membrane under Pi-starving conditions. Furthermore, the process could be monitored in wildtype cells when these were stained with FM4-64, a lipophilic dye that is endocytosed and used to mark endosomes and the vacuolar membrane (Fig. S2A). Intriguingly, the Pi-starvationinduced endocytosis and vacuolar targeting of Pho87 was prevented by the deletion of PHO4 (Fig. 2A) as well as by the deletion of the Pho4-regulated gene SPL2 (Fig. 2A andS2A). The latter was previously identified to encode a negative regulator of low-affinity Pi-transport [START_REF] Wykoff | Positive feedback regulates switching of phosphate transporters in S. cerevisiae[END_REF]. Next, we analysed Pi-starvation-induced endocytosis and vacuolar targeting of Pho90 in the wild-type and the pho4Δ, spl2 and end3 mutants using the p TEF -EGFP-PHO90 construct. In contrast to the Pho87 Pi-transporter, endocytosis of Pho90 was only prevented in the mutant strain lacking End3 and not in the strains lacking Pho4 or Spl2, indicating that the Pi-starvation-induced turnover of Pho90 occurred independent of the latter (Fig. 2B andS2B). We then examined a possible involvement of the protein kinase Rim15, another effector of the Pho85-Pho80 kinase complex [19,[START_REF] Wanke | Regulation of G(0) entry by the Pho80-Pho85 cyclin-CDK complex[END_REF]. However, this effector did not seem to play a role in the control of the low-affinity transporters, since the deletion of RIM15 did not affect the removal of Pho87 or Pho90 from the plasma membrane during Pi-starvation (Fig. 2A and2B). Recently, Spl2 was shown to interact with the N-terminal SPX-domain of Pho87 and Pho90 [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF]. This led us to analyse Pi-starvation-induced endocytosis and vacuolar targeting of Pho87Δ375N and Pho90Δ375N, representing Pho87 and Pho90 constructs lacking the Nterminal 375 amino acids. The endocytosis of Pho87 and Pho90 under Pi-starvation conditions was abrogated in the absence of their SPX-domain (Fig 2A and2B). For Pho87, this observation fits with the proposed role of Spl2 as inhibitor of low-affinity Pi-transport. For Pho90, however, the Pi-starvation-induced turnover occurs independently of Spl2 and, therefore, our data suggest that this process requires the interaction of another factor with the SPX-domain.

The SPX-domain is essential for vacuolar targeting of Pho87 and Pho90 in response to glucose starvation and rapamycin treatment. Recently, it was reported that the Pi-induced degradation of Pho84 is delayed in strains with reduced PKA activity [START_REF] Lundh | Molecular mechanisms controlling phosphateinduced downregulation of the yeast Pho84 phosphate transporter[END_REF][START_REF] Mouillon | Inhibition of the protein kinase A alters the degradation of the high-affinity phosphate transporter Pho84 in Saccharomyces cerevisiae[END_REF]. As PKA is part of a nutrient-responsive network, which also comprises the protein kinases Sch9, Rim15 and TORC1, we investigated whether other nutritional cues, besides Pi, affected the internalisation and subsequent vacuolar targeting of Pho87 and Pho90. First, to test the effect of carbon source availability, cells were transferred from ScD to Sc medium lacking a C-source for 6-8 hours. Under these conditions Pho87 and Pho90 were also endocytosed and targeted to the vacuole and this process could not be prevented by deletion of PHO4 or SPL2 (Fig. 3A and3B). Interestingly, shifting the cells from ScD medium to Sc containing galactose or glycerol already induced endocytosis of both low-affinity Pi-transporters (results not shown), letting us conclude that a downshift in the quality of the C-source is sufficient to initiate the process. Since Rim15 acts as a nutritional integrator in S. cerevisiae, we also analysed the effect of a RIM15 deletion on endocytosis and vacuolar targeting of both Pi-transporters but, similar as for Pi-starvation, the Rim15 kinase appeared not to be required (Fig. 3A and3B). Next, we challenged the cells with the TORC1-inhibiting drug rapamycin, a condition known to mimic amino acid and nitrogen starvation and to induce vacuolar targeting of different permeases in yeast, such as Gap1 and Tat2 [START_REF] Beck | The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors[END_REF][START_REF] Rohde | Nutritional control via Tor signaling in Saccharomyces cerevisiae[END_REF]. As shown, treatment of yeast cells with rapamycin also triggered endocytosis and vacuolar targeting of Pho87 and Pho90 and this was again independent of Pho4, Spl2 and Rim15 (Fig. 3A and3B). Similar results were obtained for nitrogen starvation as well (results not shown). Since the SPX-domain is essential for endocytosis and vacuolar targeting of Pho87 and Pho90 under Pi-starvation conditions, we wondered whether this domain also directs endocytosis upon glucose starvation and rapamycin treatment. Therefore, we analysed the internalisation of the Pho87Δ375N and Pho90Δ375N constructs, which revealed that the SPX-domain is indeed required to allow endocytosis of the two low-affinity Pi-transporters under both conditions (Fig. 3A and3B). Thus, despite the difference in the underlying control mechanisms and proteins involved, the internalization of the low-affinity Pi-transporters during Pi-starvation, glucose starvation or rapamycin treatment is based on the same principle, i.e. interactions of endocytosis-inducing proteins with the SPX-domain of the transporter.

The Pho90 low-affinity Pi-transporter is required for proper growth under high Pi conditions in the absence of a high-affinity Pi-transport system

The data described above focussed on the role and turnover of the low-affinity Pi-transporters under starvation conditions. In the next section, the physiological role of the low-affinity Pitransporters was investigated when sufficient amounts of Pi are present. Therefore, we evaluated the contribution of Pho87 and Pho90 to sustain growth of mutant strains lacking a functional high-affinity Pi-transport system, in relation to increasing levels of extracellular inorganic Pi. In these mutants, the high-affinity transport system was inactivated, either by deletion of PHO4, thereby inhibiting the transcription of PHO84 and PHO89, or by the combined deletion of PHO84 and PHO89. The absence of a high-affinity Pi-transport system results in a clear growth defect once the extracellular Pi levels drop below 250µM (Fig. 4A). We then additionally deleted either Pho87 or Pho90 in order to determine in more detail the contributions of each of the low-affinity Pi-transporters to sustain growth at different extracellular Pi concentrations. The pho4 pho90 and the pho84Δ pho89Δ pho90Δ strains already displayed a clear growth defect once the extracellular Pi concentrations dropped below 5 mM, while the pho4Δ pho87Δ and the pho84Δ pho89Δ pho87Δ strain displayed a growth defect only at Pi concentrations of 500µM and below (Fig. 4A). This indicates that mainly the Pho90 low-affinity Pi-transporter is important when sufficient amounts of extracellular Pi are available. These results were additionally confirmed when the growth of the different strains was further analyzed on rich YPD media and in the presence of the toxic phosphate analogue arsenate. On YPD medium, specifically the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strains exhibit growth defects when compared to the other strains examined. Consistent with the above, these defects can be suppressed by the addition of 10mM Pi, indicating again the importance of Pho90 for Pi-uptake to sustain growth (Fig. 4B). Moreover, when arsenate was added to the medium, an increased resistance to this toxic phosphate analogue was observed for both the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain (Fig. 4B). A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Discussion

The low-affinity Pi-transporters Pho87 and Pho90 are controlled by endocytosis in response to nutrient starvation Our data concerning the rapid phosphate signaling indicated that Pho4 inhibits the function of the low-affinity Pi-transporter Pho87. This inhibition appeared to be independent of PHO87 transcription, which, in agreement with previous findings [START_REF] Auesukaree | Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae[END_REF], is not regulated by Pi concentration or by major components of the PHO pathway, such as Pho4 and Pho84. This indicated that the regulation of Pho87 in function of Pi-availability must occur primarily at a posttranscriptional level and, consistently, we demonstrated that both the Pho87 and Pho90 transporters are targeted to the vacuole when yeast cells are starved for phosphate. It was previously reported that this process was likely to involve ubiquitination-induced endocytosis [START_REF] Estrella | The Rsp5 E3 ligase mediates turnover of low affinity phosphate transporters in Saccharomyces cerevisiae[END_REF], which is in agreement with our data showing a complete stabilization of Pho87 and Pho90 at the plasma membrane in Pi-starved end3 cells. Interestingly, the vacuolar targeting is impaired specifically for Pho87 in cells deleted for PHO4 or SPL2, while Pho90 is still routed to the vacuole in the pho4 strain. As the Pho87Δ375N protein, lacking its SPXdomain, also failed to be targeted to the vacuole in the absence of phosphate, our data suggest a role for Spl2 in mediating endocytosis of Pho87 via interaction with its SPX-domain [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF]. Interestingly, the Pho90Δ375N protein displayed a similar localization pattern, indicating that also for Pho90, the SPX-domain is required for its endocytosis and vacuolar targeting under phosphate starvation, although in this case the process is largely independent of the Pho4/Spl2 branch of the PHO-pathway. Hence, it can be concluded that two independent mechanisms regulate the selective vacuolar targeting of the Pho87 and Pho90 low-affinity phosphate transporters in response to depletion of phosphate from the medium, both of which are absolutely dependent on interactions with the N-terminal 375 amino acids of these proteins. Our data on endocytosis and vacuolar targeting of the low-affinity Pi-transporters clarify the results obtained in the context of phosphate signaling. Since in a wild-type strain both Pho87 and Pho90 are subject to vacuolar targeting during phosphate starvation, rapid phosphate consumption and signaling is largely depending on the high-affinity transporter Pho84 (Fig. 5). However, in a strain lacking Pho4, transcription of PHO84 is absent, rendering the cells unable to signal phosphate availability via the Pho84 transporter. However, in this strain, specifically Pho87 remains stabilized at the plasma membrane under phosphate starvation, while Pho90 is still undergoing vacuolar targeting, making that rapid phosphate consumption and signaling becomes, indeed, strictly dependent on this low-affinity Pi-transporter (Fig. 5). We also demonstrate that besides control by Pi-availability, other nutrients are able to regulate the low-affinity Pi-transporter turnover. This was documented for glucose starvation and rapamycin-treatment, the latter being a condition that mimics starvation for nitrogen and amino acids [START_REF] Rohde | Nutritional control via Tor signaling in Saccharomyces cerevisiae[END_REF]. Both conditions were found to affect both Pho87 and Pho90, and we confirm that this turnover is also driven by endocytosis [START_REF] Lundh | Molecular mechanisms controlling phosphateinduced downregulation of the yeast Pho84 phosphate transporter[END_REF][START_REF] Estrella | The Rsp5 E3 ligase mediates turnover of low affinity phosphate transporters in Saccharomyces cerevisiae[END_REF][START_REF] Wongwisansri | Disruption of histone deacetylase gene RPD3 accelerates PHO5 activation kinetics through inappropriate Pho84p recycling[END_REF]. Although the vacuolar targeting of both Pi-transporters under these conditions does not involve the Pho4/Spl2, the SPX-domains of Pho87 and Pho90 are again essential for this process. This strengthens our hypothesis that other SPX-binding factors are involved in mediating endocytosis and vacuolar targeting. Furthermore, the data indicate that both glucose starvation and rapamycin treatment induces a general vacuolar sorting response that overrules the Pi-specific feedback loops. This may allow the cell to adjust Pi-uptake not only as a function of the external Pi concentration, but also to balance the Pi-uptake in respect to other metabolic processes, thereby preventing an overflow of internal Pi-pools. As our study indicates that both Spl2 and the SPX-domain of Pho87 are required for Pistarvation-induced endocytosis of Pho87, these results fit nicely with the previously reported interaction of Spl2 with the SPX-domain of this Pi-transporter [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF]. In the same study, however, a similar interaction of Spl2 was observed with the SPX-domain of Pho90, while we did not observe a necessity for Spl2 in the endocytosis of Pho90 upon Pi-starvation, although the SPX-domain of the latter still seemed absolutely required. These results seem contradictory, but one has to keep in mind the differences in experimental approaches in our study and the previous one. The negative interaction between Spl2 and Pho90, as described by [START_REF] Hurlimann | The SPX domain of the yeast low-affinity phosphate transporter Pho90 regulates transport activity[END_REF], was seen in strains overexpressing PHO90 and/or SPL2 on rich YPD medium, which contains intermediate levels of Pi. In contrast, effects in our study were observed upon deletion of SPL2 in Pi-starvation medium. In addition, our results obtained under different nutritional conditions, as described above, indicate that other factors, besides Spl2, exist that mediate endocytosis of both Pho90 and Pho87. Consequently, overexpression of SPL2 may generate dominant effects overruling the regulation of the Pi-transporters by other factors, while deletion of SPL2 makes the regulation of Pho87 and Pho90 exclusively dependent on these factors. It is therefore very likely that Spl2 mediates the regulation of Pho90 activity under certain physiological conditions not addressed in this study. In this respect, we note that although a transcriptional regulation of SPL2 in response to Pi-levels is already documented [10,[START_REF] Wykoff | Positive feedback regulates switching of phosphate transporters in S. cerevisiae[END_REF], other modes of regulation of Spl2 function may exist which respond to different environmental cues.

Non-redundant functions for the Pho87 and Pho90 low-affinity Pi-transporters

In this study, we unveiled different situations in which either the Pho87 or the Pho90 Pitransporter is required for signaling and/or optimal growth. Indeed, we showed that in a pho4Δ background, phosphate signaling and rapid growth recovery from Pi-starvation depends specifically on the Pho87 transporter, while under conditions where sufficient amount of Pi are available, Pho90 is the most important low-affinity Pi-transporter. These transporter-specific requirements signify that Pho87 and Pho90 represent non-redundant Pitransporters which are tuned by the integration of multiple nutrient signaling mechanisms in order to adjust Pi-transport capacity to the general nutritional status of the environment. A c c e p t e d M a n u s c r i p t To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi-transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strain) at the plasma membrane, was compared with the growth of strains only containing both low-affinity Pi-transporters (the pho4Δ and the pho84Δ pho89Δ strain). Cells were grown in ScD medium and tenfold serial dilutions were spotted on Pi-starvation medium containing a specific amount of inorganic phosphate. Pictures were taken after 2-3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and tenfold serial dilutions were spotted on YPD, YPD containing an additional 10mM inorganic phosphate and/or 5mM AsO4. Pictures were taken after 3-5 days.

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118

  Biochemical Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118 A c c e p t e d M a n u s c r i p t

Figure 1 :

 1 Figure legends Figure 1: The low-affinity Pi-transporter Pho87 is essential for rapid Pi-signaling in cells lacking the transcriptional regulator Pho4The wild-type (WT) strain () and the mutants pho84 (), pho4 (), pho4 pho84 (), pho4 pho87 () and pho4 pho90 () are grown to mid-exponential phase in ScD medium after which they are transferred to Pi-starvation medium for 3 days. The cells were then analysed for (A) activation of trehalase after addition of 10 mM KH 2 PO 4 and (B) Pi consumption after re-addition of 1 mM KH 2 PO 4 to the Pi-starved cells. Figure 2: Spl2 mediates the vacuolar targeting of Pho87 but not of Pho90 under Pistarving conditions Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to Pi-starvation medium (-Pi). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture.Figure 3: The SPX-domain of Pho87 and Pho90 mediates the vacuolar targeting of both Pi-transporters during glucose starvation conditions and after rapamycin treatment Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to medium without glucose (-D). Also, the localization of both Pitransporters was monitored after treatment of exponentially grown cells for 6-8 hours with 200nM rapamycin (Rap). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture. Figure4: The Pho90 Pi-transporter is required under high Pi conditions in absence of a high-affinity Pi-transport system (A) To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi-transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strain) at the plasma membrane, was compared with the growth of strains only containing both low-affinity Pi-transporters (the pho4Δ and the pho84Δ pho89Δ strain). Cells were grown in ScD medium and tenfold serial dilutions were spotted on Pi-starvation medium containing a specific amount of inorganic phosphate. Pictures were taken after 2-3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and tenfold serial dilutions were spotted on YPD, YPD containing an additional 10mM inorganic phosphate and/or 5mM AsO4. Pictures were taken after 3-5 days. Figure5: Regulation of yeast Pi-transporters during phosphate starvation When Pi becomes limiting, the activity of the Pho85-Pho80 kinase is inhibited by Pho81, leading to nuclear translocation of the transcription factor Pho4. In the nucleus, Pho4 activates expression of typical PHO genes such as PHO84 and SPL2. Induction of Spl2 activity stimulates vacuolar targeting of Pho87, while Pho90 is targeted to the vacuole in a Spl2independent manner. Although not depicted here, we also demonstrated that under acute

Figure 2 :

 2 Figure legends Figure 1: The low-affinity Pi-transporter Pho87 is essential for rapid Pi-signaling in cells lacking the transcriptional regulator Pho4The wild-type (WT) strain () and the mutants pho84 (), pho4 (), pho4 pho84 (), pho4 pho87 () and pho4 pho90 () are grown to mid-exponential phase in ScD medium after which they are transferred to Pi-starvation medium for 3 days. The cells were then analysed for (A) activation of trehalase after addition of 10 mM KH 2 PO 4 and (B) Pi consumption after re-addition of 1 mM KH 2 PO 4 to the Pi-starved cells. Figure 2: Spl2 mediates the vacuolar targeting of Pho87 but not of Pho90 under Pistarving conditions Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to Pi-starvation medium (-Pi). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture.Figure 3: The SPX-domain of Pho87 and Pho90 mediates the vacuolar targeting of both Pi-transporters during glucose starvation conditions and after rapamycin treatment Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to medium without glucose (-D). Also, the localization of both Pitransporters was monitored after treatment of exponentially grown cells for 6-8 hours with 200nM rapamycin (Rap). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture. Figure4: The Pho90 Pi-transporter is required under high Pi conditions in absence of a high-affinity Pi-transport system (A) To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi-transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strain) at the plasma membrane, was compared with the growth of strains only containing both low-affinity Pi-transporters (the pho4Δ and the pho84Δ pho89Δ strain). Cells were grown in ScD medium and tenfold serial dilutions were spotted on Pi-starvation medium containing a specific amount of inorganic phosphate. Pictures were taken after 2-3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and tenfold serial dilutions were spotted on YPD, YPD containing an additional 10mM inorganic phosphate and/or 5mM AsO4. Pictures were taken after 3-5 days. Figure5: Regulation of yeast Pi-transporters during phosphate starvation When Pi becomes limiting, the activity of the Pho85-Pho80 kinase is inhibited by Pho81, leading to nuclear translocation of the transcription factor Pho4. In the nucleus, Pho4 activates expression of typical PHO genes such as PHO84 and SPL2. Induction of Spl2 activity stimulates vacuolar targeting of Pho87, while Pho90 is targeted to the vacuole in a Spl2independent manner. Although not depicted here, we also demonstrated that under acute

Figure 3 :

 3 Figure legends Figure 1: The low-affinity Pi-transporter Pho87 is essential for rapid Pi-signaling in cells lacking the transcriptional regulator Pho4The wild-type (WT) strain () and the mutants pho84 (), pho4 (), pho4 pho84 (), pho4 pho87 () and pho4 pho90 () are grown to mid-exponential phase in ScD medium after which they are transferred to Pi-starvation medium for 3 days. The cells were then analysed for (A) activation of trehalase after addition of 10 mM KH 2 PO 4 and (B) Pi consumption after re-addition of 1 mM KH 2 PO 4 to the Pi-starved cells. Figure 2: Spl2 mediates the vacuolar targeting of Pho87 but not of Pho90 under Pistarving conditions Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to Pi-starvation medium (-Pi). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture.Figure 3: The SPX-domain of Pho87 and Pho90 mediates the vacuolar targeting of both Pi-transporters during glucose starvation conditions and after rapamycin treatment Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to medium without glucose (-D). Also, the localization of both Pitransporters was monitored after treatment of exponentially grown cells for 6-8 hours with 200nM rapamycin (Rap). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture. Figure4: The Pho90 Pi-transporter is required under high Pi conditions in absence of a high-affinity Pi-transport system (A) To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi-transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strain) at the plasma membrane, was compared with the growth of strains only containing both low-affinity Pi-transporters (the pho4Δ and the pho84Δ pho89Δ strain). Cells were grown in ScD medium and tenfold serial dilutions were spotted on Pi-starvation medium containing a specific amount of inorganic phosphate. Pictures were taken after 2-3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and tenfold serial dilutions were spotted on YPD, YPD containing an additional 10mM inorganic phosphate and/or 5mM AsO4. Pictures were taken after 3-5 days. Figure5: Regulation of yeast Pi-transporters during phosphate starvation When Pi becomes limiting, the activity of the Pho85-Pho80 kinase is inhibited by Pho81, leading to nuclear translocation of the transcription factor Pho4. In the nucleus, Pho4 activates expression of typical PHO genes such as PHO84 and SPL2. Induction of Spl2 activity stimulates vacuolar targeting of Pho87, while Pho90 is targeted to the vacuole in a Spl2independent manner. Although not depicted here, we also demonstrated that under acute

Figure 4 :

 4 Figure legends Figure 1: The low-affinity Pi-transporter Pho87 is essential for rapid Pi-signaling in cells lacking the transcriptional regulator Pho4The wild-type (WT) strain () and the mutants pho84 (), pho4 (), pho4 pho84 (), pho4 pho87 () and pho4 pho90 () are grown to mid-exponential phase in ScD medium after which they are transferred to Pi-starvation medium for 3 days. The cells were then analysed for (A) activation of trehalase after addition of 10 mM KH 2 PO 4 and (B) Pi consumption after re-addition of 1 mM KH 2 PO 4 to the Pi-starved cells. Figure 2: Spl2 mediates the vacuolar targeting of Pho87 but not of Pho90 under Pistarving conditions Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to Pi-starvation medium (-Pi). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture.Figure 3: The SPX-domain of Pho87 and Pho90 mediates the vacuolar targeting of both Pi-transporters during glucose starvation conditions and after rapamycin treatment Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to medium without glucose (-D). Also, the localization of both Pitransporters was monitored after treatment of exponentially grown cells for 6-8 hours with 200nM rapamycin (Rap). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture. Figure4: The Pho90 Pi-transporter is required under high Pi conditions in absence of a high-affinity Pi-transport system (A) To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi-transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strain) at the plasma membrane, was compared with the growth of strains only containing both low-affinity Pi-transporters (the pho4Δ and the pho84Δ pho89Δ strain). Cells were grown in ScD medium and tenfold serial dilutions were spotted on Pi-starvation medium containing a specific amount of inorganic phosphate. Pictures were taken after 2-3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and tenfold serial dilutions were spotted on YPD, YPD containing an additional 10mM inorganic phosphate and/or 5mM AsO4. Pictures were taken after 3-5 days. Figure5: Regulation of yeast Pi-transporters during phosphate starvation When Pi becomes limiting, the activity of the Pho85-Pho80 kinase is inhibited by Pho81, leading to nuclear translocation of the transcription factor Pho4. In the nucleus, Pho4 activates expression of typical PHO genes such as PHO84 and SPL2. Induction of Spl2 activity stimulates vacuolar targeting of Pho87, while Pho90 is targeted to the vacuole in a Spl2independent manner. Although not depicted here, we also demonstrated that under acute

Figure 5 :

 5 Figure legends Figure 1: The low-affinity Pi-transporter Pho87 is essential for rapid Pi-signaling in cells lacking the transcriptional regulator Pho4The wild-type (WT) strain () and the mutants pho84 (), pho4 (), pho4 pho84 (), pho4 pho87 () and pho4 pho90 () are grown to mid-exponential phase in ScD medium after which they are transferred to Pi-starvation medium for 3 days. The cells were then analysed for (A) activation of trehalase after addition of 10 mM KH 2 PO 4 and (B) Pi consumption after re-addition of 1 mM KH 2 PO 4 to the Pi-starved cells. Figure 2: Spl2 mediates the vacuolar targeting of Pho87 but not of Pho90 under Pistarving conditions Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to Pi-starvation medium (-Pi). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture.Figure 3: The SPX-domain of Pho87 and Pho90 mediates the vacuolar targeting of both Pi-transporters during glucose starvation conditions and after rapamycin treatment Cells carrying a chromosomal P TEF -EGFP-PHO87 (A), P TEF -EGFP-PHO87375N (A), P TEF -EGFP-PHO90 (B) or a P TEF -EGFP-PHO90375N (B) construct were used to monitor the localization of the low-affinity transporters. Cells were grown exponentially (Exp) and transferred for 6-8 hours to medium without glucose (-D). Also, the localization of both Pitransporters was monitored after treatment of exponentially grown cells for 6-8 hours with 200nM rapamycin (Rap). As indicated, the wild-type cells or mutants carrying a PHO4, SPL2, RIM15 or END3 deletion were used. Over more than 300 fluorescent cells were counted and >90% of the cells exhibit the phenotypes as visualised in the picture. Figure4: The Pho90 Pi-transporter is required under high Pi conditions in absence of a high-affinity Pi-transport system (A) To evaluate the contribution of the Pho87 and the Pho90 low-affinity Pi-transporters to strains lacking a functional high-affinity Pi-transport system, the growth of strains containing only Pho87 (the pho4Δ pho90Δ and the pho84Δ pho89Δ pho90Δ strain) or Pho90 (the pho4Δ pho87Δ and pho84Δ pho89Δ pho87Δ strain) at the plasma membrane, was compared with the growth of strains only containing both low-affinity Pi-transporters (the pho4Δ and the pho84Δ pho89Δ strain). Cells were grown in ScD medium and tenfold serial dilutions were spotted on Pi-starvation medium containing a specific amount of inorganic phosphate. Pictures were taken after 2-3 days. (B) To evaluate the growth of these strains in the presence of the toxic phosphate analogue arsenate, cells were grown in YPD medium and tenfold serial dilutions were spotted on YPD, YPD containing an additional 10mM inorganic phosphate and/or 5mM AsO4. Pictures were taken after 3-5 days. Figure5: Regulation of yeast Pi-transporters during phosphate starvation When Pi becomes limiting, the activity of the Pho85-Pho80 kinase is inhibited by Pho81, leading to nuclear translocation of the transcription factor Pho4. In the nucleus, Pho4 activates expression of typical PHO genes such as PHO84 and SPL2. Induction of Spl2 activity stimulates vacuolar targeting of Pho87, while Pho90 is targeted to the vacuole in a Spl2independent manner. Although not depicted here, we also demonstrated that under acute

  Biochemical Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 Experimental procedures Strains Saccharomyces cerevisiae strains used in this study are listed in Table

	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118	A c c e p t e d M a n u s c r i p t

Table 1 : Strains used in this study Strain Genotype Reference

 1

	Biochemical Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118
	WT (BY4741)	Mata his3Δ1 leu2Δ0 ura3Δ0 met15Δ0	Y.K.O. (Invitrogen)
	pho84Δ	pho84::KANMX4	Y.K.O. (Invitrogen)
	pho4Δ	pho4::KANMX4	Y.K.O. (Invitrogen)
	pho4Δ pho84Δ	pho4::KANMX4 pho84::KANMX4	This study
	pho4Δ pho87Δ	pho4::KANMX4 pho87::KANMX4	This study
	pho4Δ pho90Δ	pho4::KANMX4 pho90::KANMX4	This study
	pho84Δ pho89Δ	pho84::KANMX4 pho89::LEU2	This study
	pho84Δ pho89Δ pho90Δ	pho84::KANMX4 pho89::LEU2
		pho90::KANMX4	This study
	pho84Δ pho87Δ pho89Δ	pho84::KANMX4 pho87::KANMX4
		pho89::LEU2	This study
	P TEF -EGFP-PHO87	Mata NatMx-P TEF -EGFP-PHO87	Hürlimann et al., 2007
	P TEF -EGFP-PHO87Δ375N	Mata NatMx-P TEF -EGFP-PHO87Δ375N	Hürlimann et al., 2009
	pho4Δ P TEF -EGFP-PHO87	pho4::KANMX4 NatMx-P TEF -EGFP-PHO87	This study
	spl2Δ P TEF -EGFP-PHO87	spl2::KANMX4 NatMx-P TEF -EGFP-PHO87	This study
	end3Δ P TEF -EGFP-PHO87	end3::KANMX4 NatMx-P TEF -EGFP-PHO87	This study
	rim15Δ P TEF -EGFP-PHO87	rim15::KANMX4 NatMx-P TEF -EGFP-PHO87	This study
	P TEF -EGFP-PHO90	Mata NatMx-P TEF -EGFP-PHO90	Hürlimann et al., 2007
	P TEF -EGFP-PHO90Δ375N	Mata NatMx-P TEF -EGFP-PHO90Δ375N	Hürlimann et al., 2009
	pho4Δ P TEF -EGFP-PHO90	pho4::KANMX4 NatMx-P TEF -EGFP-PHO90	This study
	spl2Δ P TEF -EGFP-PHO90	spl2::KANMX4 NatMx-P TEF -EGFP-PHO90	This study
	rim15Δ P TEF -EGFP-PHO90	rim15::KANMX4 NatMx-P TEF -EGFP-PHO90	This study
	end3Δ P TEF -EGFP-PHO90	end3::KANMX4 NatMx-P TEF -EGFP-PHO90	This study

© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2010 The Authors Journal compilation © 2010 Portland Press Limited

Biochemical Journal Immediate Publication. Published on 10 Dec 2010 as manuscript BJ20101118 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101118 A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2010 The Authors Journal compilation © 2010 Portland Press Limited

Acknowledgements

This work was funded by grants of the K.U. Leuven Research Fund (K.U. Leuven-BOF) and the Fund for Scientific Research-Flanders (FWO-Vlaanderen, G.0430.08). We thank IWT Vlaanderen (Agentschap voor Innovatie door Wetenschap en Technologie in Vlaanderen) for a fellowship granted to R.G. and P.D.S. We are very grateful to Prof. Dr. HC Hürlimann for providing the EGFP-tagged low-affinity Pi-transporter strains.