notch proteins are evolutionarily conserved cell - surface receptors for transmembrane ligands of the dsl family ( named after the delta and serrate ligands of drosophila ) . signaling by notch regulates a broad range of cell - fate decisions during development and various human diseases , including cancers ,
the extracellular part of notch contains 36 epidermal growth factor - like ( egf ) repeats carrying the ligand - binding region and three lin12/notch repeats ( lnrs ) that limit proteolytic cleavage of the receptor at the s2 site , and hence limit its activation .
the egf repeats are modified by two types of o - linked glycosylation , o - glucosylation and o - fucosylation ( figure 1a ) ; both are important for notch activity [ 3 - 6 ] ( figure 1a ) .
( a ) schematic representation of the drosophila notch receptor . the extracellular domain ( necd )
is composed of three lin12/notch repeats ( lnrs ) ( black ) and 36 egf - like repeats ; the egf repeats are predicted to be either o - glucosylated ( cyan ) , o - fucosylated ( yellow ) or doubly modified ( green ) adapted from .
the s2 cleavage site is indicated by an arrow . the intracellular domain ( nicd )
contains various elements involved in transcriptional activation : a ram domain , seven ankyrin repeats ( ank ) , a transactivation domain ( tad ) and a pest domain ( p ) .
( b , c ) two models for the roles of ofut1 in notch trafficking .
each model is illustrated in a schematic cell that is shown as wild type on the left and ofut1 mutant on the right , gray - shaded side .
( b ) model 1 : in the er , newly synthesized , unfolded notch ( with the necd in red and the nicd in white ) becomes properly folded ( necd in green ) and is o - fucosylated by ofut1 ( yellow diamond ) .
it is then transported to the plasma membrane and adherens junctions ( ajs , cyan ) through the golgi . in the absence of ofut1 , notch remains unfolded and is retained in the er . ( c ) model 2 : o - fucosylated notch is first transported via the golgi to the apical membrane and then transported to ajs by a transcytosis mechanism . in ofut1 mutant cells , notch goes to the membrane , is internalized and accumulates in an uncharacterized endocytic compartment .
o - fucosylation of notch is catalyzed by an o - fucosyltransferase ( pofut1 in mammals and ofut1 in drosophila )   that uses gdp - fucose as a substrate . the o - fucose residue added by this o - fucosyltransferase
can be further elongated by the addition of an n - acetylglucosamine by fringe , an egf - o - fucose 1,3 n - acetylglucosamyltransferase .
in drosophila , the activity of fringe is required for notch signaling events involved in boundary formation , in which it reduces the affinity of notch for serrate while enhancing that for delta .
given that ofut1 is thought to be the sole enzyme that o - fucosylates notch , the activity of fringe is predicted to require the o - fucosylation activity of ofut1 .
however , ofut1 has additional functions , and other catalytic and non - catalytic activities have been proposed [ 8,11 - 16 ] .
these include roles in the folding of notch in the endoplasmic reticulum ( er ) , in notch - ligand binding and in endocytic trafficking of notch .
there are several observations that support each of these models , and there has been controversy about which one is correct . a recent paper in bmc biology by kenneth irvine and colleagues ( okajima et al . )
the first model   proposes that ofut1 acts in the er , where it performs two separable functions : to o - fucosylate notch , thereby modulating notch - ligand interaction , and to promote the correct folding of the extracellular domain of notch ( a non - catalytic function ) .
it partially co - localizes with er markers and acts , at least in part , in the er : the carboxy - terminal extremity of ofut1 contains a lys - asp - glu - leu ( kdel)-like motif that is dispensable for its catalytic activity but is required for both er retention and function .
the notch protein accumulates in intracellular compartments marked by er markers , and knockdown of ofut1 using double - stranded rna in cultured cells inhibits the secretion of a soluble version of the notch extracellular domain ( necd ) .
this activity of ofut1 does not require its o - fucosylation activity because expression of a mutant version of ofut1 demonstrated to be catalytically dead ( ofut1 ) mostly rescues the notch localization defects .
in addition , ofut1 and ofut1both bind the necd , and expression of ofut1 in cultured cells can increase both the amount and the ligand - binding activity of secreted necd .
consistent with this proposed o - fucosylation - independent activity , notch localizes at the cell cortex in gdp - mannose 4,6-dehydratase ( gmd ) mutants , which contain no gdp - fucose , indicating that non - o - fucosylated notch exits the er . together , these results led to a simple model ( model 1 , figure 1b ) in which ofut1 acts as an er chaperone to promote the proper folding of the egf repeats of notch , thereby ensuring its correct cell - surface localization and ligand - binding activity .
the second model   proposes that ofut1 acts at two post - exocytosis steps in the trafficking of notch : it regulates both the endocytosis of notch   and its transcytosis from the apical plasma membrane to the adherens junctions ( ajs ) .
first , ofut1 is required for the proper distribution of notch at ajs . in ofut1 mutant cells
, notch does not accumulate at ajs but instead accumulates into intracellular ' dots ' that co - localize with er markers only poorly .
in addition , surface - staining experiments indicate that a low level of notch is also present at the surface of ofut1 mutant cells .
these data were interpreted   to suggest that ofut1 is not required for the er exit and cell - surface delivery of notch .
second , ofut1 is required to regulate the endocytic trafficking of notch , as monitored by antibody uptake .
anti - notch antibodies are internalized in ofut1 mutant cells , albeit at a lower level than in wild - type cells , but fail to accumulate in endosomes .
it is therefore proposed that notch accumulates in an uncharacterized endocytic compartment in ofut1 mutant cells . finally , the second model is supported by the observation that a lower level of intracellular notch is detected in gmd mutant cells than in ofut1 single - mutant or ofut1 , gmd double - mutant cells .
this suggests that the endocytic activity of ofut1 does not depend on its catalytic activity .
in addition , the failure of notch to localize at ajs in gmd mutant cells is interpreted in the light of a speculative model of the transcytosis of notch to suggest that ofut1 acts in a catalysis - dependent manner to regulate the transcytosis of notch .
we feel that this transcytosis model needs further experimental validation , so we will not detail the possible role of ofut1 in this process further .
we note , however , that this proposed role of ofut1 in the transcytosis of notch suggests that the o - fucosylation activity of ofut1 is required for fringe - independent signaling events .
together , these observations suggest a model ( model 2 , figure 1c ) in which ofut1 regulates the endocytic trafficking of notch at a post - internalization step so that notch accumulates in an undefined endocytic compartment in the absence of ofut1 activity .
thus , these two models offer two different interpretations of the cellular basis of the ofut1 mutant phenotype : in model 1 , the reason notch does not signal is because it is trapped in the er , whereas in model 2 , it is because notch accumulates in an undefined endocytic compartment with a low level of ( inactive ) notch at the surface .
one implication of model 2 is that a fraction of ofut1 must escape er retention and act in the endocytic pathway .
consistent with this possibility , experiments using cultured cells indicate that a fraction of ofut1 is secreted , interacts with the extracellular part of notch at the cell surface , and is internalized in a notch - dependent manner .
in addition , the notch localization defects seen in cells treated with ofut1 double - stranded rna can be rescued by adding ofut1-containing conditioned medium .
whether this happens in vivo is not clear . indeed , ofut1 acts in a cell - autonomous manner to regulate the localization of notch , arguing that secretion is not important in this regulation .
moreover , the observed rescue of the ofut1 knockdown phenotype by extracellular ofut1 could also be consistent with model 1 , given that endocytosed ofut1 may be transported back into the er .
so , how can we distinguish between the two models ? clearly , answering the following questions would help .
does notch reach the cell surface in the absence of ofut1 activity ? where does notch accumulate in ofut1 mutant cells ? is the o - fucosylation activity of ofut1 required for proper notch localization and activity ? or is it only required for fringe - dependent signaling ?
okajima et al .   have addressed these issues and provide compelling evidence in favor of model 1 .
okajima et al .   have shown that the o - fucosylation activity of ofut1 is required for fringe - dependent but not for fringe - independent signaling events .
they found that embryos lacking both maternal and zygotic contributions of gmd develop into larvae , as fringe mutant embryos do .
the complete loss of ofut1 activity results in a strong phenotype mimicking a loss of notch activity that is rescued to larval viability by the expression of ofut1 .
these data show that the o - fucosylation activity of ofut1 is dispensable for notch signaling in the embryo .
this conclusion is entirely consistent with the observation that the activity of fringe is dispensable in the embryo .
it is therefore clear that non - fucosylated notch can reach the cell surface and can signal .
the role of the o - fucosylation activity of ofut1 during wing imaginal disc development led to identical conclusions .
although the loss of ofut1 activity in clones leads to phenotypes mimicking a loss of notch activity , expression of ofut1rescued notch receptor activity in these ofut1 mutant cells and led to phenotypes mimicking a loss of fringe activity .
this confirms that non - fucosylated notch can signal , and further indicates that the fucosylation activity of ofut1 is required only for fringe - dependent signaling events .
this therefore implies that the transcytosis step of model 2 , which was proposed to be dependent on ofut1 catalytic activity , is not essential for notch signaling . because the controversy over the exact role of ofut1 is in part due to methodological differences between the various studies and also to technical limitations in subcellular localization analysis , okajima et al .  
also re - examined the localization of notch in ofut1 mutant cells using a detergent - free cell - surface staining protocol .
a striking difference in surface staining was observed between wild - type and ofut1 mutant cells .
this convincingly shows that notch is not present at detectable levels at the surface of mutant cells .
this contradicts the results obtained by sasaki et al . , who used a different protocol to assay the presence of notch at the cell surface of ofut1 mutant cells .
whether the difference in protocols accounts for these opposite conclusions remains to be addressed experimentally .
the second piece of evidence in support of a cell surface accumulation of notch in ofut1 mutant cells comes from antibody uptake experiments showing that anti - notch antibodies can be internalized by ofut1 mutant cells .
however , as discussed by okijama et al . , the low level of antibody uptake can probably be accounted for by the fluid - phase uptake of anti - notch antibodies by live ofut1 mutant cells , followed by the specific retention of internalized antibodies by notch accumulating intracellularly .
together , the published data are best interpreted as concluding that notch does not reach the cell surface in ofut1 mutant cells .
notch was shown to partially co - localize with four different er markers that , in fact , show only partial co - localization among themselves .
thus , the only partially overlapping distribution of er markers may explain the poor co - localization of notch with the two er markers seen by sasamura et al . .
therefore propose that notch accumulates in ofut1 mutant cells in the er , which is a heterogeneous organelle .
accordingly , notch should co - localize better with the sum of the signals of the different er markers .
it is clear that the o - fucosylation of notch is primarily required for fringe - dependent signaling events and that ofut1 acts non - catalytically to regulate the exit of notch from the er . thus , ofut1 probably acts as a chaperone in the er to promote the proper folding of the extracellular domain of notch , as described in model 1 .
although the catalytic and non - catalytic activities of ofut1 can be experimentally uncoupled , it is attractive to speculate that the o - fucosylation activity of ofut1 participates in the quality - control mechanism that ensures that only properly folded notch exits the er . further analysis of the trafficking of non - fucosylated notch , produced for instance by gmd mutant cells , would help address this issue .
 the precise role of the o - fucosyltransferase ofut1 in notch - receptor trafficking has remained controversial . 
 a recent study sheds new light on the non - catalytic activity of ofut1 and provides further evidence that ofut1 acts as a chaperone in the endoplasmic reticulum . 