extfieldsofstudy: Biology Medicine

text:
HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease.

Introduction
Herpes simplex viruses (HSV) are the leading cause of genital herpes worldwide, the most common infection associated with neonatal encephalitis, and a major co-factor for HIV infection, thus underscoring the urgency to develop novel prevention strategies [1]. Notably, the epidemiology of genital herpes may be changing as recent studies indicate that HSV-1 accounts for a significant proportion of new infections, particularly in the developed world [2,3]. Identifying new approaches to prevent infection by both serotypes requires an understanding of the pathways required for the establishment of primary and recurrent infection and the cellular factors usurped by the viruses to promote infection.
Preventing HSV entry has proved difficult, reflecting the complexity of this process, which involves interactions between several viral envelope glycoproteins and cellular receptors and activation of calcium signaling pathways. Both serotypes (HSV-1 and HSV-2) initiate infection by binding to heparan sulfate moieties on syndecan proteoglycans [4,5,6,7]. Glycoprotein D (gD) then engages one of several entry receptors, most commonly nectin-1 or herpes virus entry mediator (HVEM) [8,9]. Studies with human epithelial cells indicate that these viral-cell interactions trigger the release of calcium (Ca 2+ ) near the plasma membrane, which is followed by activation of the inositol triphosphate receptor, resulting in the rapid release of endoplasmic reticulum (ER) Ca 2+ stores [4,10]. This release of ER stores requires the concerted activities of glycoproteins B, D, and heterooligomers of H and L and blockade of the Ca 2+ response prevents viral entry.
Recent work suggested that another cellular protein also may play a role in HSV entry. Porcine renal epithelial cells, which are naturally resistant to HSV entry, were rendered fully susceptible following transfection with a cDNA encoding human hfl-B5 [11,12,13,14]. The B5 protein was found to be ubiquitously expressed on multiple human cell lines and a synthetic 30-mer peptide containing the sequence found in the C-terminus of B5 inhibited HSV infection at a step following viral attachment [14]. Recent genetic studies demonstrate that B5 is identical to the sequence that encodes for subunit m of eukaryotic initiation factor 3 (eIF3m) [15]. Building from these observations, we sought to further explore the role B5 (eiF3m) plays in HSV infection of human cells and whether it could provide a target for the development of novel prevention strategies.

Silencing of B5 inhibits HSV infection
CaSki (human cervical epithelial) cells were transfected with siRNA targeting B5, nectin-1, an established entry co-receptor, or as a control, HVEM, an alternative co-receptor that is not expressed at detectable levels on CaSki cells [4]. Silencing resulted in reductions of 80-95% in protein and RNA expression by Western blot and quantitative real time PCR (qRT-PCR), respectively, compared to cells transfected with siHVEM (Figs 1A and B) or with a non-specific control siRNA (not shown). Silencing was specific, as transfection with siB5 had no impact on nectin expression and, conversely, transfection with siNectin had no effect on B5 expression. To determine whether silencing impacted HSV infection, CaSki cells were transfected with siB5, siNectin, or siHVEM. The transfected cells were infected with HSV-2(G) or HSV-1(KOS) 48 h post-transfection and viral plaques were quantified. Transfection with siB5 or siNectin reduced HSV-2 plaque formation by 98.660.8 and 86.563.5% respectively and HSV-1 plaque formation by 68615 and 66611%, respectively, relative to cells transfected with a non-specific control siRNA, whereas silencing of HVEM had little or no effect (Fig. 1C). Transfection with each individual siRNA targeting B5 also resulted in a reduction in B5 protein expression and concomitant inhibition of HSV-2 plaque formation, although siB5#1 was the least efficient (Fig. 1D). Importantly, transfection with siB5 was not cytotoxic and did not trigger any significant interferon response (not shown). Together, these findings support a role for B5 in HSV infection.

B5 is not required for HSV entry
Several parallel strategies were adopted to determine whether B5 silencing interfered with viral entry. VP16 is a virion tegument protein delivered to the nucleus following HSV entry and thus serves as a surrogate marker of viral entry. CaSki cells were transfected with nectin-1, control, or B5 specific siRNA, and then 48 h later, synchronously infected with HSV-2(G) (moi 1 pfu/cell). Forty-five minutes post-shift of the temperature to 37uC, cells were harvested and nuclear extracts were prepared and evaluated for . CaSki cells were transfected with siHVEM (control), B5 or nectin-1-specific pools of siRNA and 48 h post-transfection, immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab or anti-nectin mAb and then stripped and probed for b-actin expression. (B) Western blots were scanned for protein expression (odu). In parallel studies, RNA was extracted from transfected cells and analyzed by qRT-PCR for expression of B5 and nectin. Results are presented as protein or RNA expression as a percentage of cells transfected with siHVEM and are means 6 sd obtained from 3 different experiments. (C) The transfected cells were infected with HSV-2(G) or HSV-1(KOS) (48 h post-transfection) and the ability to support viral plaque formation determined by counting plaques after 48 h incubation by immunostaining. Results are presented as pfu/well as a percentage of pfu/well formed in cells transfected with non-specific control siRNA and are means + sd from 2 independent experiments conducted in quadruplicate. (D) Cells were transfected independently with each siB5, with the pool of three siRNAs or with siHVEM (control) and 48 h post-transfection immunoblots of cell lysates were prepared and probed with anti-B5 polyclonal Ab and b-actin and relative expression determined after scanning the blots. Results are presented as odu relative to cells transfected with siHVEM. In parallel, the transfected cells were infected with HSV-2(G) and the ability to support viral plaque formation determined by counting plaques after 48 h incubation by immunostaining. Results are presented as pfu/well as percentage of pfu/well formed in cells transfected with siHVEM and are means 6 sd from a representative experiment conducted in duplicate. doi:10.1371/journal.pone.0011829.g001 the presence of VP16 by preparing Western blots; expression of nuclear protein histone-1 was included as a control. As anticipated, silencing of nectin-1 significantly blocked the nuclear transport of VP16 by 78615% (p,0.01, t-test), consistent with a block to viral entry, whereas silencing of B5 had no impact on VP16 nuclear delivery relative to cells transfected with control siRNA ( Fig. 2A).
We also applied confocal microscopy to directly monitor the intracellular delivery of viral capsids. CaSki cells were transfected with each of the siRNAs and 48 h later, synchronously infected (moi 5 pfu/cell) with purified KVP26GFP, an HSV-1 variant expressing a VP26-GFP fusion capsid protein, for which the viral envelope was also labeled with a fluorescent long-chain carbocyanine dye, DiI [4,16]. This enabled discrimination between bound viral envelopes (red) and intracellularly delivered capsids (green). Notably, as previously reported, the GFP fluorescence in the capsid is only visualized after viral de-envelopment when the viral envelope is labeled with DiI [4]. The cells were stained with EZ-Link Sulfo-NHS-Biotin to detect plasma membranes (blue) before infection and were fixed 15 minutes after entry. Bound viral envelopes (red) were readily detected in all of the transfectants. However, while viral capsids (green) were observed in the majority of cells transfected with siHVEM (control) or B5-specific siRNA, they were seen in fewer than 6% of cells transfected with nectin-1 siRNA (Fig. 2B).
The block to entry in response to nectin-1, but not B5 silencing, was further confirmed by comparing the intracellular Ca 2+ response to HSV-2(G) (moi 5) by live confocal microscopy [4]. We previously reported that HSV triggers the release of intracellular Ca 2+ , which promotes viral entry. Viral envelopes (DiO labeled, green) were readily detected bound to the plasma membrane (blue) of cells 10 minutes after the temperature reached 37uC, but release of intracellular Ca 2+ (crimson) was only observed in the control or siB5 transfected cells, not the siNectin-1 Figure 2. Silencing of nectin, but not B5, prevents HSV entry. (A). CaSki cells were transfected with siNectin, siHVEM or siB5 and then 48 h later, synchronously infected with HSV-2(G) and 45 min after the temperature shift to 37uC, nuclear extracts were prepared and analyzed for presence of viral VP16 by Western blot; expression of histone-1 was included as a control. The bar graph depicts expression of VP16 relative to histone-1; results are representative of three independent experiments. (B). Cells were transfected with siRNA and then 48 h post-transfection, infected with DiIenvelope labeled KVP26GFP and 15 min later, fixed and stained for plasma membrane (blue) and analyzed by confocal microscopy. Images are representative of 3 independent experiments: 100 cells were counted and the bar graph depicts percentage of cells expressing bound virus (red envelope) and viral capsids (green). (C). Representative 3-dimensional images obtained by live confocal microscopy of transfected cells, stained for plasma membrane (blue), loaded with Calcium Crimson and then synchronously infected with DiO-envelope labeled HSV-2(G) (green). Images shown were obtained 10 min after being placed in a 37uC temperature-controlled chamber. The bar graph on the right depicts relative intensity obtained from scanning 10 cells per field of vision from two independent experiments. doi:10.1371/journal.pone.0011829.g002 transfectants (Fig. 2C). No intracellular Ca 2+ response was detected in cells transfected with siNectin as late as 30 minutes following infection (not shown). Additionally, phosphorylation of focal adhesion kinase, a cellular response to viral entry that promotes transport of the viral capsids to the nuclear pore, was observed following HSV infection of cells transfected with siB5, but not cells transfected with siNectin (not shown) [17]. Together, these findings demonstrate that B5 contributes to HSV infection at a step downstream of viral entry and nuclear transport.

B5 plays key role in the translation of HSV proteins
The finding that the sequence for B5 is identical to that of eIF3M [15] suggested that B5 might participate in the initiation of viral protein translation. To address this possibility, CaSki cells were again transfected with siB5 or siHVEM as a control, infected (moi 1 pfu/cell) with HSV-2(G) 48 h post-transfection, and then nuclear or whole cell lysates prepared at different times postinfection (pi) were evaluated for viral protein expression by Western blots. Incoming VP16 was readily detected in nuclear extracts prepared 45 min post-HSV-2 infection in cells transfected with siHVEM (control) and siB5, reflecting the absence of any block to viral entry or nuclear transport. However, 4 h pi, little or no newly synthesized VP16 could be detected in cells transfected with siB5 compared to cells transfected with siHVEM (Fig. 3A). Comparable results were obtained for other viral proteins including thymidine kinase and gB (not shown). A marked reduction in expression of viral immediate early genes (ICP0, ICP4 and ICP27) was observed in whole cell lysates harvested 4 h pi from cells infected with HSV-1(KOS) (Fig. 3B).
To determine whether the reduction in protein expression reflects a block in transcription or translation, transfected cells were infected (moi 1 pfu/cell) with HSV-2(G) and then RNA was isolated at different times pi and analyzed for the expression of ICP0, ICP4 or gB by qRT-PCR. As an additional control, cells were infected with virus and then treated with 100mg of cycloheximide, an inhibitor of protein synthesis. Neither transfection with siB5 nor treatment with cycloheximide prevented transcription of ICP0 or ICP4 (Fig. 4). However there was a marked reduction in gB transcripts, which presumably reflects the requirement for ICP0 and ICP4 proteins, which are transcriptional transactivators of late viral gene expression [18,19]. These results suggest that siB5 does not block immediate early gene expression at transcription, but blocks the translation of these viral proteins.  Cross-linking of protein-RNA complexes indicates that B5 interacts with viral mRNA To explore whether B5 interacts directly with viral mRNA, cells were infected with HSV-2(G) (2 pfu/cell) and 3, 5 or 7 h pi, the cells were treated with formaldehyde to cross-link RNA-protein complexes, and then the complexes were immunoprecipitated with a polyclonal antibody to B5. Western blots confirmed that B5 was found exclusively in the pellet (not shown). RNA was extracted from both the supernatant and pellet and evaluated for the presence of viral transcripts and RPLPO by qRT-PCR. The ICP4 and ICP0 transcripts were detected almost exclusively in the B5immunoprecipitated pellet with peak expression detected at 3 and 5 hours pi, respectively. In contrast, gB transcripts were primarily detected in the supernatant with peak expression 7 h pi and RPLPO transcripts, a ribosomal host cellular gene, were detected in both the supernatant and pellet (Fig. 5A).
In parallel studies, the complexes were subjected to Western blotting with antibodies to ICP4 or gB for HSV-2 and ICP0 for HSV-1; ICP0 was not evaluated for HSV-2 as the antibody is serotype specific. ICP4 and ICP0 were detected in the pellet as early as 3 h pi, whereas gB was detected primarily in the supernatant 5 and 7 h pi and RPLPO was detected in both the supernatant and pellet (Fig. 5B). These findings are consistent with the qRT-PCR results (Fig. 5A) and suggest that B5 interacts with immediate early gene transcripts to promote their translation.

B5 is required for translation of viral ICP0 and ICP4
The observation that silencing of B5 led to a reduction in HSV immediate early proteins without affecting mRNA expression suggests that B5 is required for translation of the viral immediate early proteins. Specifically, silencing could interfere with mRNA processing, such as polyadenylation, nuclear export, the association of RNA with ribosomal subunits or polysomes and/or translation of the mRNA to protein. To address these possibilities, additional experiments were conducted. Transfected cells were infected with HSV-2(G) (3 pfu/cell) and 3 h pi, cells were harvested and nuclear and cytoplasmic fractions isolated. Gol-gin97 and histone-1 were detected in the cytoplasmic and nuclear fractions, respectively, confirming the purity of the fractionation. B5 and ICP4 were detected in the cytoplasm of cells transfected with control siHVEM, but expression was markedly reduced or absent in cells transfected with siB5 (Fig. 6A). Polyadenylated mRNA was purified from the total isolated RNA and from the cytoplasmic and nuclear fractions and visualized in a formalde- Figure 5. Viral RNA and immediate early proteins associate with B5 in RNA-protein complexes. (A) Cells were infected with HSV-2(G) and 3, 5, or 7 h pi, the cells were treated with 0.04% of methanol-free formaldehyde to cross-link RNA-protein complexes. The complexes were immunoprecipitated with a polyclonal rabbit anti-B5. and RNA was extracted from the supernatant and pellet and analyzed for the expression of viral transcripts and RPLPO. Results are shown for the time pi at which peak expression of viral transcripts was detected (3 h for ICP4, 5 h for ICP0 and 7 h for gB) and are presented as the ratio of viral transcript/RPLPO in each fraction to the viral transcipts/RPLPO in the total RNA-protein complex from infected cells prior to immunoprecipitation (input). Expression of RPLPO is presented as the ratio of RPLPO in each fraction to RPLPO in the total RNAprotein complex from uninfected cells prior to immunoprecipitation (input). (B) The supernatant and pellet from the immunoprecipitated complexes isolated at 0, 3, 5, and 7 h pi were evaluated for the expression of ICP4 (HSV-2), gB (HSV-2), ICP0 (HSV-1) or RPLPO by Western blots. Blots are representative of results obtained in at least 3 independent experiments. doi:10.1371/journal.pone.0011829.g005 hyde denaturing gel. No differences in polyadenylated RNA were detected (not shown) and similar proportions of ICP4 polyadenylated mRNA were detected by qRT-PCR in both the nuclear and cytoplasmic fractions in cells transfected with siHVEM or siB5 (Fig. 6B). Together, these results indicate that B5 silencing had no deleterious impact on polyadenylation or nuclear export of RNA.
To determine if silencing of B5 impacted the ability of viral transcripts to associate with ribosomal subunits or polysomes, polysome fractions were isolated from cells that had been transfected with siHVEM (control) or with siB5 by centrifugation through 10-50% sucrose gradients. The UV absorbance pattern of fractions derived from cells that had been transfected with siB5 differed from the pattern observed in siHVEM transfected cells, consistent with B5 playing a role in the formation of ribosomal subunits (Fig. 7A, left). RNA was isolated from each fraction and analyzed for expression of GAPDH by reverse-transcription and PCR and, in parallel studies, the proteins in each fraction were analyzed by Western blot for cytolasmic poly(A)-binding protein 1 (PABP), which is a general translation factor for mRNA, and for B5. GAPDH and PABP were present across the gradient from both siHVEM and siB5 transfected cells, although modest differences in relative amounts expressed in various fractions were observed (Fig. 7A, right). B5 protein was detected predominantly in fractions 7-9 of the siHVEM control cells, coinciding with the 80S ribosomal subunit, and was absent in the siB5 transfected cells.
Parallel studies were conducted following infection with HSV-2. Consistent with published studies, the gradient profile following viral infection in the control (siHVEM) cells showed reduced levels of the ribosomal subunits compared to uninfected cells, presumably reflecting viral-induced shutoff of host protein synthesis (Fig. 7B, left) [20]. The gradient profile was further altered in the siB5-transfected cells, possibly reflecting the combined effects of silencing and viral infection. GAPDH was again distributed across the gradient in fractions isolated from infected cells transfected with either siHVEM or siB5, but ICP4 transcripts were only detected in the fractions isolated from the control (siHVEM) cells and were found primarily in fractions 6-8, which coincides with the fractions containing B5 protein (Fig. 7B, right). Similarly, Western blots demonstrated that ICP4 polypeptides could only be detected in the fractions isolated from control, but not siB5 transfectants (Fig. 7B, right)). Taken together, these results suggest that in the absence of B5, ICP4 transcripts fail to associate with ribosomal subunits, resulting in a block in translation.

B5 silencing has a greater impact on HSV than VSV or HIV infection
Studies were extended to other cell types and other viruses to explore whether B5 also played a major role in protein synthesis and infection. Silencing of B5 inhibited HSV infection of HaCAT cells (human keratinocyte) and murine fibroblasts (not shown). Moreover, there was a reduction in ICP0 protein expression in siB5 transfected Jurkat-TAT-CCR5 cells following HSV-1 infection (Fig. 8A, left). In contrast, silencing of B5 in these cells had a non-significant impact on HIV infection as measured by p24 in culture supernatants (p = 0.2) (Fig. 8A, right). Transfection of CaSki cells with siB5 had more modest effects on VSV (p = 0.03) relative to the inhibitory effects on HSV (p = 0.002) (Fig. 8B).

Silencing of eIF2a, but not B5, is cytotoxic
Most studies of interactions between HSV and host cell initiation factors have focused on eIF2a because of its established role in host defense. A major cellular response to viral infection (or other stress) is to inactivate eIF2a by phosphorylation. HSV overcomes this host response through the actions of at least two viral proteins, ICP34.5 and Us11, which cooperate to restore eIF2a pools [21]. While eIF2, a heterotrimer composed of three subunits, is essential for protein synthesis, the precise role eIF2a, the regulatory subunit, plays in HSV protein translation has not been established. Thus, we compared the impact of silencing eIF2a and B5 on viral infection. Transfection of CaSki cells with sieIF2a efficiently silenced its expression by ,80%; siB5 had little or no effect on eIF2a expression (Fig. 9A). Transfection with either siB5 or sieIF2a reduced ICP4 expression as detected by Western blots of infected cellular lysates 4 h pi (Fig. 9B) and also inhibited viral plaque formation by at least one log (not shown). However, there was a marked difference in the effects of silencing on cell viability. Silencing of eIF2a reduced cell viability by ,50% 3 days post-transfection, whereas siB5 had little effect (Fig. 9C). The cytotoxic effects observed with sieIF2a were comparable to those observed with the detergent N-9 [22]. These results suggest that B5 may provide a safer and more specific target to block HSV protein synthesis.
B5 provides a target for prevention of primary or recurrent HSV Currently, the primary modality for treatment or suppression of HSV infections is acyclovir, which interferes with viral DNA replication. We compared the effects of acyclovir and B5 silencing on viral infection alone, or in combination. Cells were transfected with siB5 or siHVEM and then challenged with serial dilutions of HSV-2(G) in the absence or presence of 100mg/ml acyclovir and the viral plaques determined. Treatment with acyclovir or transfection with siB5 each independently reduced viral plaque formation by ,2-logs and, when combined, resulted in ,3-log reduction (Fig. 10A), indicating that the two interventions target different steps in the viral life cycle. To explore whether targeting of B5 could block the amplification of virus that had reactivated, we used an explant co-cultivation model. Sacral ganglia were removed from vaginally infected mice and co-cultured for two weeks with CaSki cells that had been transfected with siB5 or control siRNA; viral amplification was monitored by collecting a portion of the culture supernatants and assaying for the presence of virus by plaque assay. Little or no virus was detected in culture supernatants harvested from co-cultures of sacral ganglia and B5silenced cells, whereas .10 4 pfu/ml were recovered from cocultures of ganglia with cells transfected with control siRNA in two independent experiments (not shown).
To further explore the potential safety of targeting B5 to reduce primary or recurrent HSV, the effects of B5 silencing on the overall pattern of host protein synthesis were examined by metabolic labeling the cells 72 h post-transfection. Little change in host cell protein expression was observed (Fig. 10B, left); the efficiency of silencing was confirmed by Western blots (Fig. 10B,  right). These findings, coupled with the lack of cytotoxicity in B5 transfected cells (Fig. 9C) indicate that B5 is not essential for host cell protein translation and support the notion that B5 could provide a novel target for antiviral therapeutic strategies.

Discussion
Productive HSV infection requires the translation of viral proteins and is associated with the selective inhibition of host protein synthesis. While substantial work has focused on delineating how HSV triggers the shutoff of host protein synthesis, far less is known about the molecular mechanisms that promote viral protein synthesis. The results of this study begin to address this gap. Specifically we found that B5, which has been recently identified as eIF3m, plays a key role in promoting the initial translation of HSV immediate early proteins, ICP4 and ICP0. The pivotal role B5 (eIF3m) plays in initiating the synthesis of HSV immediate early proteins may be relatively specific, as silencing of B5 had little effect on HIV, more modest effects on VSV, and little or no impact on host cellular proteins including eIF2a and nectin-1. The exact determinant that regulates which viral or cellular transcripts will interact with B5 requires further study.
Currently at least 12 eIFs, composed of at least 29 distinct subunits, have been identified [23]. The largest mammalian initiation factor, eIF3, plays an essential role in translation by binding directly to the 40S ribosomal subunit to recruit mRNAs that bear a methylated guanosine cap at the 59-end and promote formation of the 43S preinitiation complex. Mammalian eIF3 consists of 13 non-identical subunits, eIF3a through eIF3m; subunit m was the most recently discovered [15]. The functions of the individual subunits in human cells are not yet well characterized. In the yeast Saccharomyces cerevisiae, eIF3 consists of a core complex of five stoichiometric subunits (eIF3a, -b, -c, -g, and -i), plus a non-stoichiometric subunit, eIF3j. In mammalian cells, the corresponding homologues also may constitute a ''core'' complex to which the other mammalian subunits bind and regulate eIF3 activity. The finding that silencing of B5 had little effect on cell viability and that it did not adversely impact expression of the cellular proteins studied is consistent with the notion that eIF3m is not an essential core element. However, further studies are needed.
The notion that distinct subclasses of eIF3 complexes, containing different combinations of core and non-core subunits, may regulate specific subsets of mRNA has been suggested in studies with fission yeast, where biochemically distinct eIF3 complexes defined by the PCI domain proteins eIF3e and eIF3m associate with different sets of mRNAs [24]. Tandem mass spectrometry demonstrated that eIF3m is likely to be on the periphery of the eIF3 complex, and possibly accessible to viral mRNA [15]. Additionally, time-resolved spectroscopy has shown that the B5 C-terminus forms a reversible structure that might allow it to interact with proteins, viral RNA or RNA-protein complexes [25]. Notably, a recent study found that eIF3m may also play a role in regulating caspase 9 responses to apoptotic stimuli [26].
Previous studies have explored interactions between HSV proteins and eIF4 and eIF2a. Specifically, ICP0 and ICP6 stimulate the assembly of eIF4F complexes, which facilitate subsequent viral protein synthesis [27,28]. However, these interactions must occur downstream of the interactions between HSV immediate early mRNA and eIF3m (B5). The ability of HSV to antagonize eIF2a phosphorylation, which would prevent formation of the 80S ribosomal complex and thus block viral protein synthesis, has been extensively studied. Results of the current studies are consistent with the role of eIF2a in viral protein translation, as silencing reduced HSV gene expression and plaque formation (Fig. 9). However, silencing of eIF2a is toxic to cells and thus would not provide a target for antiviral therapies.
The observation that both acyclovir and siB5 markedly inhibit HSV infection by different mechanisms supports the notion that targeting of B5 could provide a novel strategy to prevent primary and recurrent HSV infections. This is critical, not only because of the morbidity associated with HSV infection itself, but because of the synergy between HSV and HIV. In recently completed clinical trials, suppressive acyclovir therapy failed to reduce the risk for HIV acquisition [29]. One possible explanation for this comes from recent work showing the persistence of activated immune cells in sequential biopsies of HSV-2 lesions from patients on suppressive antiviral therapy [30]. Acyclovir inhibits viral replication, but does not prevent viral reactivation or block viral protein expression, and therefore may not prevent the recruitment and activation of HSV-specific T cells. Results obtained in this work suggest that B5 silencing might prevent the amplification of reactivating virus and possibly limit the recruitment and activation of T cells, which may serve as HIV targets.
The discovery that B5 plays a key role in promoting the synthesis of HSV immediate early proteins was serendipitous. The original observation that B5 may contribute to HSV infection came from studies with porcine cells that are resistant to HSV entry and did not address the translation of viral proteins. Transfection of the porcine cells with a human cDNA expressing B5 rendered the cells fully susceptible to HSV [11]. However, in ongoing work, we found that B5 does not play a direct role in mediating entry into the porcine cells, but acts indirectly and modulates the expression of porcine nectin, rendering it accessible to bind viral gD (work in progress). Notably, we observed no Figure 9. Silencing of eIF2a, but not B5, is cytotoxic. (A) Cells were transfected with HVEM, eIF2a or B5 specific siRNA or no transfection (media) and 48h later, evaluated for the expression of eIF2a and b-actin (control) by Western blot; blots are representative of 2 independent experiments. (B) Cells were treated with media or transfected with siB5, sieIF2a, or siHVEM and then 48h later, infected with HSV-2(G) and 4h pi, cell lysates were prepared and evaluated for expression of viral ICP4 and b-actin; a representative blot from 3 independent experiments is shown (left). The blots were scanned and results are presented as odu relative to cells treated with media (mean + sd from 3 experiments). (C). Cells were grown on 96-well plates, transfected with siB5 or sieIF2a or treated with 0.1% N-9, Effectene, or untreated and then quadruplicate wells were evaluated daily for 5 days for cell viability using an MTT assay. Results are presented as percent viability relative to untreated cells and are means 6 sd obtained from 2 independent experiments. doi:10.1371/journal.pone.0011829.g009 changes in expression of human nectin-1 following B5 silencing. In conclusion, the current studies demonstrate that B5 (eiF3m) plays a major role in promoting the translation of HSV immediate early proteins and may be a novel target for prevention strategies.

Cells and Viruses
CaSki cells (human cervical epithelial cell line) were obtained from American Tissue Culture Collection (ATCC CRL 1550) and were passaged in DMEM supplemented with 10% fetal bovine serum. Jurkat-Tat-CCR5 cells, a T-cell line transfected with the HIV-1 tat gene and the CCR5 coreceptor, were cultured in complete RPMI supplemented with 250 mg/ml hygromycin B (for Tat selection) and 500 mg/ml geneticin (for CCR5 selection) as previously described [31]. HSV-2(G), HSV-1(KOS), and HSV-1(KVP26GFP), which contains a green fluorescent protein (GFP)-VP26 fusion protein [16] were prepared from infected Vero cell cultures (ATCC CCL81) and viral stocks were stored at 280uC. HIV-1 BaL was grown in PM-1 cells and stored at 280uC after filtration through 0.2 mm filters (Millipore, MA). VSV-Indiana was grown on Vero cells.

Viral labeling and purification
To label viral envelopes, Vero cells were infected with wild-type viruses or KVP26GFP virus (,0.001 pfu/cell) and after 48 h, the infected cells were lysed and then incubated with lypophilic tracers DiO or DiI (1 mM) (Molecular Probes, Invitrogen Valencia, CA) for 10 min at room temperature before purification on sucrose gradients [4].

Viral infection and VP16 assays
Transfected or control cells were infected with serial dilutions of virus for 1 h at 37uC, inoculum removed, and cells overlaid with medium in the absence or presence of acyclovir (100mg/ml) (American Pharmaceutical Partners, Schaumburg, IL) for 48 h. Plaques were counted by immunoassay using an anti-human IgG antibody peroxidase conjugate (Calbiochem) [32]. For synchronous infections, cells were precooled and exposed to virus at 4uC for 4 h to allow binding. Unbound virus was removed by washing, and the cells were transferred to 37uC to allow viral penetration for 45 min. For VSV, CaSki cells that had been transfected with siRNA 48 h earlier were infected with serial dilutions of VSV, overlaid with 1% methylcellulose, fixed after 24 h with methanol, and plaques counted after staining with Giemsa.
To examine transport of VP16 to the nucleus, nuclear extracts were prepared 45 min after infection and analyzed by Western blots as described previously [10]. To examine viral protein or gene expression, whole cell lysates were prepared and protein expression examined by Western blots or RNA extracted for qRT-PCR (below). In select experiments, cycloheximide (100mg/ml, C-7698, Sigma-Aldrich) was added to the overlay media for the entire time of infection as a control for inhibition of protein synthesis.
Total RNA extraction and real-time quantitative RT-PCR (qRT-PCR) Total RNA was extracted using an Absolutely RNA Miniprep kit (Stratagene, La Jolla, CA). RNA (500ng) was reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). Real-time PCR amplification was performed using an Applied Biosystems 7900HT Real-Time PCR System and analyzed using the sequence detector software. The reactions were performed using the TaqManH Gene Expression Master Mix and the probes for human nectin-1 (PVRL1) (Hs00161050_m1), HVEM (TNFRSF14) (Hs00187058_m1), B5 (GA17) (Hs00272235_m1), interferon alpha (IFNA1) (Hs00256882_s1) and RPLPO (4310879E) were obtained from Applied Biosystems. Quantification was normalized against the number of RPLPO transcripts in the same RNA extracts.
To evaluate expression of HSV genes, primer and probe sequences were designed using Primer Express (Applied Biosystems) software and synthesized by Applied Biosystems as 206Mix.

Confocal Microscopy
Cells were grown on glass coverslips in 12-well plates, transfected with siRNA as described above, and, if indicated, 24 or 48 h post-transfections, synchronously infected with DiI-labeled HSV-1(KVP26GFP). To label plasma membranes, the cells were stained for 30 min with EZ-Link sulfosuccinimidobiotin (EZ-Link Sulfo-NHS-Biotin) reagent (0.1 mM; Pierce Chemical, Rockford, IL), which reacts with primary amines on cell-surface proteins before infection, fixed with 4% paraformaldehyde solution (Electron Microscopy, Hatfield, PA) at the indicated times pi, and the biotinylated cells were reacted with Alexa Fluor 350conjugated streptavidin (1:1000 dilution; S11225, S11249; Invitrogen, Carlsbad, CA). Images were acquired by laser confocal microscope ZeissLive/DuoScan equipped with an oil immersion objective 6361.4. Images were captured in an optical slice of ,0.5 mm with appropriate filters, Alexa Fluor 488, and GFP were excited using the 488-nm line of a krypton/argon laser and viewed with a 505-to 530-nm band pass mm. AlexaFluor 360 were excited with 405-nm diode laser and collected with 420-to 475 nm filter, Alexa 546 were excited using 543-nm helium/neon laser and collected with a 575-to 655 filter. All images were captured using the multitrack mode of the microscope to decrease cross talk of fluorescent signals. Z-sections were captured in an optical slice of 0.5 mm.
Live image microscopy was performed to examine calcium signaling in response to HSV. Cells were grown in glass bottom culture dishes 35 mm (product no. P35G-1.5-10-C; MatTek corporation, Ashland, MA) and labeled with EZ-Link and 350conjugated streptavidin (1:1000 dilution; S11225, S11249; Invitrogen). The cells were then loaded with Calcium Crimson (C-3018, 2.5mg/ml, Invitrogen) for 4 h at 4uC, and infected with DiO-labeled virus for 4 h at 4uC. Cells were washed three times with PBS to remove extracellular Ca 2+ and unbound virus, overlaid with 25 mM HEPES buffer, and placed into a temperature-regulated 37uC environmental chamber in a ZeissLive/DuoScan confocal microscope fitted with a 10061.4 oil objective. Images were acquired 10 min after the dishes were placed in the chamber. Image analysis was conducted using the LSM confocal software package (Carl Zeiss, Inc.) and quantification of intensity staining with image J software (National Institutes of Health, Bethesda, MD). Three-dimensional (3-D) images were generated using the Volocity 4 confocal software (Improvision, Lexington, MA).

RNA-protein complexes and immunoprecipitation
CaSki cells were grown on 25cm 2 flasks, transfected with siRNA at 800pM per flask using the Effectene transfection reagent (QIAGEN, Valencia, CA) and 48h post-transfection, the cells were infected with HSV2(G) (moi 2 pfu/cell). Three, 5 or 7 h pi, the cells were treated with 0.04% of methanol-free formaldehyde (RT 15680, Electron Microscopy sciences, Hatfield, PA) to cross-link RNA-protein complexes or with PBS as a control and immunoprecipitated with a polyclonal rabbit anti-B5 antibody in RIPA buffer (Sigma-Aldrich) supplemented with complete protease inhibitors (Roche Diagnostics) as described [33]. The RNA was purified from the supernatant and the precipitated complexes (pellet) and analyzed by qRT-PCR using primers for ICP4, ICP0, gB or RPLPO. Quantifications were normalized per input RNA. Enrichment was defined as the ratio of normalized number of viral transcripts in each fraction relative to transcripts in the input (nonimmunoprecipitated). Immunoblots were also prepared and analyzed for viral proteins or RPLPO.

Polyadenylated RNA extraction
Subconfluent CaSki cells were transfected with 800pmol/well (T25 dish) of siHVEM or siB5 and then 48 h post-transfection were infected with HSV2(G) (3 pfu per cell). Three hours pi, cells were harvested and dissolved into iso-hi-pH buffer [10mM Tris pH 8.4, 140mM NaCl, 1.5mM MgCl 2 , 0.5% NP-40] supplemented with 10mM VRC (S1402S, New England BioLabs, Ipswich, MA). Nuclei were pelleted for 10 min at 1500 rpm, washed twice in isohi-pH and once in iso-hi-pH supplemented with 0.1 volume of Tween-deoxycholate solution, pelleted through a 2.2 M sucrose pad for 30 min at 16,000 rpm, and washed again in iso-hi-pH. To examine the purity of isolated cellular fractions, Western blots were performed using golgin97 and histone H1 as markers of cytoplasm and nuclear fractions, respectively. Total RNA was purified from the total cellular lysates, cytoplasmic and nuclear fractions by TRIzol, and polyadenylated mRNA was subsequently isolated from the total RNA preparations using Absolutely mRNA TM purification kit (400806, Stratagene) and visualized by formaldehyde denaturing gels. Expression of ICP4 was detected by qRT-PCR.

Isolation of polysomes
Subconfluent CaSki cells were transfected with 800pmol/well (T25 dish) of siHVEM or siB5 and then 48 h post-transfection were left uninfected or were infected with HSV2(G) (3 pfu per cell) for 3 h. Both the uninfected and infected cells were trypsinized and the cell pellets were rinsed twice with ice-cold PBS and resuspended in gradient buffer [50mM Tris-HCL (pH 7.5), 80mM KCl, 5mM MgOAc, 2 mM DTT, 1% sucrose] containing 1 unit/ ml of RNase inhibitor (RNAsin, N2611, Promega, Madison, WI) and supplemented with complete protease inhibitors (Roche). Cells were lysed by sonication, subjected to centrifugation (16,000 rpm for 10 min at 4uC) and then layered on top of an 11-ml linear 10-50% sucrose gradient. Samples were centrifuged at 27,500 rpm in a Beckman SW41 ultracentrifuge rotor for 4 h. Gradients were fractionated from the bottom (1 ml fractions) and analyzed for PABP, B5 or ICP4 by Western blotting. Alternatively, the RNA in each fraction was purified with TRIzol LS reagent (Life Technologies, Gaithersburg, MD) followed by phenol-chloroform extraction and ethanol precipitation and dissolved into 10 ml of RNAase free water. The fractions were assayed for absorbance at 254 nm with a Nano Drop spectrophotometer. To evaluate for ICP4 transcripts, 2ml of RNA recovered from each fraction were subjected to reverse transcription and then the cDNA was amplified with primers specific for ICP4 or GAPDH (ICP4: forward 59-GTACGTCAGCGGGGAGCC-39 and reverse 59-GTCGCCGTCGAAGCCCTC-39; GADPH: forward 59-GCATGGCCTTCCGTGTCCC C-39 and reverse 59-TGGCAGGGACTCCCCAGCAG-39. DNA was visualized by electrophoresis in 1% agarose.

HIV infection and p24 assay
Jurkat-TAT-CCR5 cells were infected with HIV-1 BaL (4ng of p24 per 10 4 cells) for 2 hours and then washed and overlaid with RPMI growth media. Culture supernatants were collected and viral replication was assessed using a p24 enzyme-linked immunosorbent assay (ELISA; p24 antigen capture assay kit; NCI-Frederick Cancer Research and Development Centre, AIDS Vaccine Program).


text:
Proteomic insights into an expanded cellular role for cytoplasmic lipid droplets.

Cytoplasmic lipid droplets (CLDs) are cellular structures composed of a neutral lipid core surrounded by a phospholipid monolayer of amphipathic lipids and a variety of proteins. CLDs have classically been regarded as cellular energy storage structures. However, recent proteomic studies reveal that, although many of the proteins found to associate with CLDs are connected to lipid metabolism, storage, and homeostasis, there are also proteins with no obvious connection to the classical function and typically associated with other cellular compartments. Such proteins are termed refugee proteins, and their presence suggests that CLDs may serve an expanded role as a dynamic protein storage site, providing a novel mechanism for the regulation of protein function and transport.

signifi cant quantities of ether-linked glycerolipids have also been found ( 12 ). The phospholipid species in the monolayer refl ect the composition of the endoplasmic reticulum (ER) ( 12,13 ), despite some differences observed in fatty acid composition ( 13 ).
Although CLDs can be found in virtually every cell type, some intrinsically produce more (e.g., cells involved in lipid metabolism such as adipocytes and hepatocytes, mammary epithelial cells during pregnancy and lactation ( 14 ), and fat body tissue from Drosophila embryos) ( 15,16 ). Disease phenotypes can also dramatically alter the total numbers and size of CLDs (e.g., type II diabetes, obesity, neutral lipid storage diseases like Chanarin-Dorfman Syndrome, liver diseases such as steatohepatitis or liver cirrhosis, and cardiovascular diseases such as atherosclerosis). For cells in culture, CLD formation can be stimulated by adding fatty acids bound to albumin and/or cholesterol to promote the production of CLDs ( 17 ).
Abstract Cytoplasmic lipid droplets (CLDs) are cellular structures composed of a neutral lipid core surrounded by a phospholipid monolayer of amphipathic lipids and a variety of proteins. CLDs have classically been regarded as cellular energy storage structures. However, recent proteomic studies reveal that, although many of the proteins found to associate with CLDs are connected to lipid metabolism, storage, and homeostasis, there are also proteins with no obvious connection to the classical function and typically associated with other cellular compartments. Such proteins are termed refugee proteins, and their presence suggests that CLDs may serve an expanded role as a dynamic protein storage site, providing a novel mechanism for the regulation of protein function and transport . Supplementary key words adipose differentiation related protein • adipophilin • PAT-family proteins • protein sequestration • mass spectrometry Previously, cytoplasmic lipid droplets (CLDs) were considered to be relatively simple passive energy storage structures. However, recent studies reveal the intriguing possibility that CLDs are dynamic cellular structures and may play a role in protein sequestration as a mechanism to regulate the protein abundance and function within cells as well as lipid homeostasis. Furthermore, there is evidence that CLDs may also play a role in the regulation of membrane traffi cking. This review seeks to analyze the contribution of proteomic studies to the growing body of knowledge over the last decade. [Please refer to recent comprehensive reviews for the cell biology and formation of CLDs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11).] CLDs are composed of a neutral lipid core surrounded by a phospholipid monolayer. Lipids in the neutral core are primarily triacylglycerols and sterol esters, although sample. For shotgun approaches, choosing a multidimensional protein analysis involving several steps of chromatography may lead to a greater number of identifi cations versus single phase chromatographic runs at the cost of requiring increased analysis time and a more complicated experimental approach ( 36 ). Altering chromatographic conditions (solid phases, mobile phases, gradients, heated columns, and column lengths) can affect the peptides ionized for detection by tandem MS. Mass spectrometers have different capabilities in terms of resolution, ionization method, and analysis speed (duty cycle) that affect the depth of profi ling that can be achieved.
One very important factor to consider when comparing CLD proteomes from different studies is the choice of database search algorithms and databases. This topic has been addressed in recent studies aimed at improving standardization between methods of proteomic analysis and database searches (37)(38)(39). False discovery rate is a measure of the statistical signifi cance analysis as it applies to mass spectrometric peptide identifi cation and this subject is given a more in-depth treatment in some recent papers (40)(41)(42).

PROTEOMIC STUDIES
The application of proteomic analyses to CLDs within the last decade have revealed a great deal about the proteins associated with CLDs in a variety of mammals, insects, and fungi. The following sections summarize the major fi ndings in each study and are organized according to cell/tissue origin.

SACCHAROMYCES CEREVISIAE
Saccharomyces cerevisiae was used for two proteomic studies and the results are summarized in supplementary Table  I . CLDs from wild-type and deletion yeast strains were analyzed by 1D gel/MS analysis by Athenstaedt et al. (18) and proteins identifi ed were those involved in sterol formation, lipid synthesis and lipid metabolism, enzymes, proteins associated with the ER, and several proteins with unknown functions.
A second study showed a relationship between yeast CLDs and peroxisomes ( 19 ). Proteins identifi ed using proteomics were localized using electron microscopy (EM), immunoEM, and fl uorescence microscopy in intact S. cerevisiae cultured in either control glucose (2%) or in oleic acid. In cells cultured with oleic acid, there were increased numbers of CLDs and peroxisomes. Although only observed in a small number of cases (3.2% of the cell sections where both CLDs and peroxisomes were visible), the peroxisomes were found to extend "pexopodia" into adjacent lipid droplets. Formation of pexopodia was correlated to the nutritional state of the cells, with pexopodia formation being promoted when cells were forced to scavenge fatty acids from CLDs. CLDs from these oleic acid cultures were fractionated and a shotgun approach was employed for proteomic analysis. Proteins identifi ed were Two general categories of proteomic platforms have been used to analyze fractionated CLDs. The fi rst approach separates proteins according to molecular weight using gel electrophoresis (1D or 2D) followed by in-gel protease digestion. The peptides are extracted from the gel and separated by chromatography followed by MS analysis ( 29 ). The other approach is sometimes termed "shotgun" proteomic analysis, whereby complex protein samples are digested to peptides in solution, and the peptides are then separated by chromatography followed by MS analysis. To date, most of the proteomic analyses of CLDs have used the fi rst approach ( 14,16,18,(20)(21)(22)(23)(24)(25)28 ) whereas only a few have used either a combined approach ( 15,27 ) or a stand-alone shotgun proteomic approach ( 19,26 ).
All of the proteomic analyses have utilized chromatographic separations of peptides at ambient temperatures. Recently, it was demonstrated that elevated temperatures dramatically improve the recovery of hydrophobic proteins from reverse-phase chromatographic columns ( 30,31 ). The hydrophobic nature of the neutral lipid core of CLDs implies that proteins embedded and/or attached to CLDs are generally hydrophobic in nature, or at a minimum have one or more hydrophobic domains. Proteins are thought to attach to lipid droplets through surface interactions or through helical hairpins of hydrophobic peptides that extend into the lipid core of the LD (32)(33)(34).
[Note: Hydrophilic proteins have also been observed to be embedded within the lipid core ( 35 ).] Therefore, the incorporation of this analytical modifi cation during proteomic analyses may have the potential to improve the comprehensiveness of the current CLD proteomes.
The issue of minimal overlap of proteins identifi ed from different samples derived from CLDs poses a signifi cant challenge in developing a comprehensive CLD proteome. Actual differences between the proteins associated with CLDs in different samples is one reason that proteins may be detected in one sample but not another, but this is not the only reason for disparities in proteins detected. Other reasons may include differences in metabolic state of cells used to isolate CLDs in addition to differences in sample preparation, differences in proteomic methods used (i.e., gel based methods versus shotgun methods), limitations of instrumentation, and differences in searching algorithms or databases used for protein identifi cation.
Sample preparation and the choice of proteomic method can affect which proteins are observed from different samples. Like other biological samples, CLDs contain a wide range of protein abundances, and proteins most likely to be identifi ed using proteomic methods are those present at high abundance, whereas low abundance proteins are more diffi cult to detect. Within proteomic approaches, there are sample preparation factors that may lead to differences in the proteins detected. For example, in gel-based proteomic methods covered in this review, some investigators analyzed entire 1D-SDS PAGE gel lanes cut into small pieces ( 16,21 ) versus analyzing only gel bands stained in 1D-SDS PAGE gels ( 18,20,22,24,25,28 ), with the goal of identifying more proteins from a CLD tions, and class B, which was composed of proteins identifi ed only once. In class A, 60 proteins were identifi ed as being common to all four genotypes with smaller numbers of proteins identifi ed that overlapped just one or two genotypes, with a very small number of proteins identifi ed that were genotype specifi c. The lack of large qualitative differences between these genotypes indicates that genotype characteristics producing leanness or obesity of adult Drosophila may not be refl ected in the lipid droplet subproteome of the Drosophila embryos at this early stage of their development. One exception to this fi nding was the observation of the Regucalcin protein in adp 60 (obesity predisposed genotype), a homolog of the vertebrate protein senescence marker protein-30 (SMP30) that has been shown in mice to affect cellular lipid droplets, body weight, and lifespan. Other proteins identifi ed repeatedly in different CLD preparations are proteins involved in fatty acid/lipid metabolism, enzymes modifying short-and long-chain fatty acids, membrane traffi cking proteins (Rabs 2, 5, and 6), chaperones, and ER-resident proteins.

Mammalian cells in culture
CHO K2 cells. Analysis of Chinese hamster ovary (CHO) K2 cells using 1D gel/MS analysis identifi ed many novel CLD proteins ( 20 ). Cultured CHO K2 cells grown in normal media were homogenized, then fractionated and washed repeatedly to remove contaminants from cytosol or membranes. Samples were imaged using EM and light microscopy. Thirty-seven proteins were identifi ed and can be categorized into four groups: lipid metabolism, membrane traffi cking, signaling, and miscellaneous. The full protein list can be found incorporated into supplementary Table III. Lipid metabolism is the largest grouping, with 13 proteins (35% of proteins identifi ed). Of these, only four [S3-12, adipose differentiation related protein or adipophilin (ADRP), PDI, and BIP] were previously identifi ed as lipid droplet associated proteins. Several of these proteins were related either by sequence or function to yeast proteins identifi ed by Athenstaedt et al. ( 18 ) Proteins in the lipid metabolism category were typical of those found in other mammalian CLD studies. The membrane traffi cking protein group was composed of 11 proteins (30% of identifi ed proteins). The membrane traffi cking proteins identifi ed include numerous members of the Rab family of small GTPases (Rabs 1, 2, 5c, 7, 10, 11a, 11b, 14, and 18). The signaling protein group was small, containing only three of the total proteins identifi ed (8%). Western blot analyses were carried out to verify the results obtained from the MS experiments. A second set of CHO K2 cells were incubated with oleic acid to promote CLD formation. The biggest change observed in the cells exposed to oleic acid came from a marked increase of ADRP, along with the identifi cation of an additional gel band ( ف 40 kDa) containing CGI-58 protein.
The second study to use CHO K2 cells was published in 2007 by Bartz et al. ( 12 ) and emphasized several goals. classifi ed into three categories: 1 ) proteins that had previously been identifi ed as LD proteins by Athenstaedt et al. ( 18 ), 2 ) mitochondrial, peroxisomal and/or ER-associated proteins, and 3 ) cytosolic proteins. The peroxisomal proteins identifi ed were all matrix components that are involved in the ␤ -oxidation of oleic acid. Many mitochondrial proteins were identifi ed and these were further classifi ed as either matrix proteins, matrix proteins localized to nucleoids, or inner/outer membrane or nucleoid proteins. Numerous resident proteins from the ER as well as membrane traffi cking proteins, nuclear proteins, chaperones, enzymes, plasma membrane-associated proteins, and two histones, and numerous cytosolic proteins were observed.

DROSOPHILA
Two recent studies were carried out in Drosophila and their results are summarized in supplementary Table II. Ceremelli et al. ( 15 ) characterized CLDs isolated from the embryos of Drosophila followed by shotgun proteomic analysis to identify 127 proteins that appeared in more than three separate studies. [Note: 453 additional proteins were identifi ed in only one study (397 proteins) or two studies (56 proteins)]. Proteins were classifi ed into several groups. Lipid metabolism proteins, membrane traffi cking proteins (Rab 8, and Rab 11), heat shock proteins, and other organellar proteins were observed. Surprisingly, several histone proteins (H2A, H2B, and H2Av) were identifi ed repeatedly and at high abundances within these CLD preparations. Further orthogonal studies demonstrated that during the fi rst few hours of embryonic development, the protein levels of H2A and H2B increased but not in direct proportion to the number of nuclei, indicating that the embryos have enough H2A and H2B to package chromatin for thousands of nuclei at the time of fertilization. Importantly, Ceremelli et al. ( 15 ) demonstrated that the large deposits of maternal histones can be stored on CLDs and rapidly and effi ciently transferred to chromatin as they are needed, thereby providing a new embryo with a source of preexpressed histones for rapid embryonic development. Furthermore, by storing the proteins in an inactive form on CLDs, retardation of embryonic growth that might result from the unregulated presence of large quantities of free histones is also avoided.
The second proteomic study of Drosophila using 1D gel/ MS analysis, by Beller et al. ( 16 ), focuses on lipid droplets from Drosophila fat body tissue to examine the lipid droplet subproteome from Drosophila predisposed to obesity ( adp 60 and induced Lsd-2:EGFP ) or leanness ( Lsd-2 51 ) . This group identifi ed 248 proteins, some that were labeled using GFP to localize the proteins within intact cells. EM studies of the cells and Western blot analyses were performed. Of the total 248 proteins identifi ed, 60 proteins were identifi ed in all four genotypes studied, 127 were found in wildtype larvae, 137 proteins were found in adp 60 larvae, 153 were found in induced Lsd-2:EGFP , and 159 were found in Lsd-2 51 mutant larvae. Proteins identifi ed were separated into two classes: class A, composed of proteins that were identifi ed reproducibly in separate lipid droplet prepara-lipolytically stimulated adipocytes included PAT-family protein adipophilin, lipid metabolism proteins, cytoskeletal proteins, membrane traffi cking proteins (several Rab GTPases) and several proteins of unknown function.
The second study of 3T3-L1 adipocyte CLDs was undertaken with the goal of exploring proteins involved in lipid droplet biogenesis using 2D gel/MS analysis ( 23 ). The 3T3-L1 cells were cultured for 10 days after differentiation before cells were homogenized and CLDs were enriched by centrifugation. Although many of the proteins observed by this group were identifi ed in previous proteomic studies, several new proteins were identifi ed and included a lipid metabolism protein, four membrane traffi cking proteins, and several proteins of unknown function/localization [a 54.10 kDa protein PRP19 (homolog of S. cerevisiae PSO4) that was selected for further study].
The 504 amino acid protein Prp19p is encoded by the murine Prp19 gene (mPrp19). In S. cerevisiae and humans, Prp19p has functions in DNA combination and error prone repair and previously had not been observed to have functions outside the nucleus ( 23 ). This protein acts as a scaffold for protein-protein interactions in the nucleus, and the authors suggest that Prp19p may play a role in the formation or stabilization of CLDs through the ability to make protein-protein interactions. Stable 3T3-L1 cell lines were developed to either overexpress mouse Prp19 (pCDNA3.1-mPrp19 ) or downregulate mouse Prp19 (p-mPrp 19RNAi). RNAi downregulation of Prp19p decreased intracellular fat accretion, thereby repressing lipid droplet biogenesis and expression of SCD1, DGAT-1, GPAT, perilipin, and S3-12, but it did not affect other genes important to adipocyte differentiation. Unlike perilipin, Prp19p does not appear essential for cAMP-and hormone sensitive lipase-dependent lipolysis pathways.

Mammalian cells from tissue
Mouse mammary epithelial cells. An early study of mammalian tissues focused on CLDs obtained from mammary epithelial cells from the inguinal mammary glands, livers, and milk taken from the same lactating animal. Milk fat globules (MFGs) and milk fat globule membranes (MFGMs) were isolated from the mouse milk ( 14 ). Proteomic analysis was performed on these four sample types using 2D gel/ MS analysis. Discontinuous sucrose gradients were used for subcellular fractionation of CLDs, MFGs, and MFGMs, followed by washing to de-enrich for proteins not strongly associated with the lipid-rich CLDs, MFGs, or MFGMs. This study employed orthogonal methods (EM and Western blotting) to verify the enrichment of the CLDs and deenrichment of other proteins.
The mammary CLD proteins identifi ed through tandem MS were largely similar to those found in the MFGM sample (see supplementary Table III for full protein list). Only four proteins were common to the liver and mammary CLDs (ADRP, FABP, and probable contaminant serum proteins transferrin and albumin). Proteins observed in the mammary sample included chaperones, ER-resident proteins, and cytoskeletal proteins in addition to proteins known to be associated with CLDs (ADPH, FABP). The The fi rst goal of this study was to identify more of the proteins from CLDs by analyzing both the unstained and stained regions of the gels. The second goal was to identify phosphorylated proteins isolated from HeLa cell CLDs. The third goal of this study was to examine purifi ed lipid droplets incubated in cytosol either in the presence or absence of GTP ␥ s to determine whether lipid droplets are capable of recruiting effector proteins from the cytosol.
The complete proteome of CLDs from the cultured CHO K2 cells showed 125 proteins with 70 proteins newly recognized as being associated with CLDs (see supplementary Table III). The global analysis by 1-D gel/MS analysis of the CHO K2 proteome revealed numerous proteins previously identifi ed as CLD-associated proteins either in CHO K2 CLDs ( 20 ) or in CLDs from other cell types ( 22, 24-28, 43, 44 ).
Numerous novel CLD proteins identifi ed by Bartz et al. included lipid metabolism proteins, novel membrane traffi cking proteins [including many Rab family proteins (Rabs 19b, 21, 24, 33b, 34, 35, and 41)] as well as some other small GTPase proteins (ArfGAP1 and ARL10C), vesicle transport-related proteins, signaling proteins, cytoskeletal proteins, endoplasmic reticulum proteins, and lysozyme proteins, chaperones, and one protein each from the Golgi and mitochondria. Three unknown proteins were identifi ed, and these were classed as hypothetical protein DKFZp434P0316, KIAA0242 protein, and p25.
Phosphorylation is the control mechanism used by some proteins involved in lipolysis such as perilipin ( 45,46 ) or hormone sensitive lipase ( 47 ). Bartz and coworkers used the 1D gel/MS proteomic platform to analyze CLDs isolated from HeLa cells to identify phosphorylation sites on acetyl CoA carboxylase 1, ADRP, NADH dehydrogenase precursor, phospholiase A2 (group IVA), Rab 5, Rab 8, and adipose triglyceride lipase (TTS-2.1 or iPLA2-zeta). Furthermore, this group also identifi ed some additional small GTPases and cofactors that may be selectively recruited to CLDs by incubating purifi ed CLDs with cytosol in the presence or absence of GTP ␥ s in CHO K2 cells.
Mouse 3T3-L1 adipocyte cells. Lipid droplets from cultured adipocytes of the mouse strain 3T3-L1 were analyzed in two studies and both are summarized in supplementary  Table III ( 22,23 ). The initial study by Brasaemle et al. ( 22 ) compared the proteomes of CLDs isolated from basal state adipocytes versus those that were lipolytically stimulated using isoproterenol, an agonist of ␤ -adrenergic receptors, and isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, using 1D gel/MS analysis. In addition to proteomic analyses, this study also used fl uorescence microscopy to confi rm the localizations of identifi ed proteins within the cells. PAT family proteins perilipin, S3-12, and TIP47 were found on both control and lipolytically stimulated cells but adipophilin (ADRP) was found selectively on droplets that were lipolytically stimulated. Lipid metabolism proteins, ER-resident proteins, cytoskeletal proteins and several chaperone proteins were found in both preparations of CLDs from control and lipolytically stimulated cells. Proteins identifi ed only in CLDs from identifi ed using 1D gel/MS analysis, with 23 CLD proteins identifi ed from the HCV-expressing cells and 15 CLD proteins identifi ed from the control cells. Thirteen of the proteins identifi ed in the control cells overlapped with proteins identifi ed in the HCV-expressing cells. Among the proteins observed using 1D gel/MS analysis were two PAT family proteins, seven proteins involved in lipid metabolism, membrane traffi cking proteins (Rabs 1A, 1B, 5C, and 7), three proteins involved in RNA metabolism and binding, one mitochondrial protein, and several proteins of unknown functions or that do not fi t into other categories.
Using the shotgun approach, Sato and coworkers identifi ed 36 total CLD proteins, with 27 CLD proteins found in the HCV-expressing cells and 24 CLD proteins found in the control cells with 15 CLD proteins observed in both cell types. Twenty-two CLD-associated proteins overlapped with proteins identifi ed using 1D gel/MS analysis. Proteins identifi ed via the shotgun approach but not identifi ed using 1D gel/MS analysis were fi ve lipid metabolism proteins, three additional membrane traffi cking proteins (Rabs 8, 10, and 11), two RNA metabolism/binding proteins and proteins that did not fi t within these categories. Interestingly, both the 1D gel/MS analysis and shotgun analysis showed that RNA metabolism/binding proteins were only found in the HCV-expressing cells and these were not observed in the control cells. Several proteins were observed to be associated with CLDs for the fi rst time in this study (sterol carrier protein 2-related form, 58.85K, FABP5, and apoptosis-inducing factor homologous mitochondrion-associated inducer of death).

Human epithelial and MDCK cultured cell lines. Cultured cell lines of human epithelial cells (UAC and A431) and
Madin Darby Canine Kidney (MDCK) cells were studied in 2004 by Umlauf et al. ( 24 ) to identify CLD-associated proteins, and specifi cally to examine stomatin, due to its biochemical and topological similarity with caveolins (results included in supplementary Table III). Caveolins are known to associate with CLDs ( 22,(48)(49)(50). Transfected cells were cultured to express GFP-tagged stomatin, and immunofl uorescence was used to localize stomatin to CLDs within the cultured cells. Western blot analyses were used to identify proteins and enrichment of CLD-associated proteins in the CLD samples. Proteomic 1D gel/MS analysis was employed to study the A431 squaemous epithelial carcinoma cells.
Through immunofl uorescence microscopy, stomatin was found to associate with CLDs when expressed exogenously at high levels or expressed endogenously in the presence of oleic acid. If vesicular transport was blocked, the levels of endogenous stomatin were increased causing an increase in association with CLDs. Cycloheximide treatment caused stomatin dissociate from CLDs and stomatin subsequently associated with acidic vesicles. Time lapse video microscopy of live cells was used to show that CLDs interact with small vesicles and occasionally CLDs show high motility.
Proteomic analysis of CLDs from the A431 cells revealed 33 CLD-associated proteins. Studies were carried out on MFGM samples contained all the proteins identifi ed in the mammary CLD fraction, but contained additional proteins involved in lipid metabolism and milk proteins, along with some apolipoproteins.

Rat liver cells after traumatic liver injury. In 2006, Turró et al. ( 27 ) studied the CLDs produced in liver tissue after traumatic liver injury (a partial hepatectomy) in Sprague
Dawley rats. A dramatic increase of fat was observed in the cytoplasm 24 h after a partial hepatectomy. Enriched CLDs were analyzed using a combined approach of 2D gel/MS analysis and shotgun proteomics resulting in 24 and 27 proteins identifi ed, respectively. Approximately 30% of the total proteins observed were identifi ed by both methods (see supplementary Table III for a full protein list). Four ER-resident proteins, two transport-related proteins, four cellular enzymes, and one protein of unknown function [0610006F02Rik protein-later renamed methyltransferase like protein 7B (Uniprot: Q6UX53)] were uniquely identifi ed using the 2D gel/MS analysis approach. Five proteins not previously associated with CLDs were identifi ed using shotgun analysis, including an ER-resident protein, three cellular enzymes and one protein of unknown function [610006F02Rik (methyltransferase like protein 7B)]. The 0610006F02Rik protein (methyltransferase like protein 7B) was subsequently expressed as a recombinant GFP-tagged protein and expressed in COS cells. Fluorescence microscopy localized the protein to CLDs, and to a smaller extent, the ER.

Human cells in culture
Human cultured liver cell lines. Two studies have analyzed CLDs from two cultured human liver cell lines, hepatocyte cell line HuH7 ( 25 ) and hepatoma cell line HepG2 ( 26 ). Results are included in supplementary Table III. Fujimoto et al. ( 25 ) examined CLDs isolated from HuH7 hepatocyte cells by subcellular fractionation using sucrose gradients followed by 1D gel/MS analysis. Seventeen proteins were identifi ed with two of those being proteins that had been previously observed to associate with CLDs (ADRP and TIP47). The most abundant proteins identifi ed were ADRP and two other lipid metabolism proteins. A phospholipid binding protein and fi ve lipid metabolizing enzymes were identifi ed. Four ER-resident proteins and one cytosolic protein were identifi ed in the CLD samples but no other organellar proteins were identifi ed within the CLD samples. GAPDH, two acyl-CoA synthetases, lanosterol synthestase, and an SDR family protein had been observed to be associated with CLDs of S. cerevisiae previously, indicating that there may be functional conservation of these proteins within eukaryotic species ( 18 ). The role of CLDs in lipid metabolism is critical to maintaining lipid homeostasis and, not surprisingly, proteins involved in lipid metabolism are observed ubiquitously in the CLDs. These include two 17-␤ hydroxysteroid dehydrogenases, acetyl-CoA carboxylase ␣ , CGI-49 protein, CGI-58 protein (lipase modulator), dehydrogenase/reductase (SDR family) member 1, lanosterol synthase, long-chainfatty-acid-CoA ligases, NADH-cytochrome b5 reductase 3, patatin-like phospholipase domain-containing protein 2 [also called adipocyte triglyceride lipase (TTS-2.1) (iPLA2 )], protein disulfi de-isomerase A3, squalene epoxidase, and NAD(P)-dependent steroid dehydrogenase-like protein. These proteins play a variety of roles in lipid synthesis (long-chain-fatty-acid-CoA ligases, lanosterol synthase, squalene epoxidase), lipid homeostasis (long-chain-fatty-acid-CoA ligases), and lipid degradation (patatin-like phospholipase domain-containing protein 2, CGI-58 ( 64 ).) Aside from the PAT family proteins and those involved in lipid metabolism, several proteomic studies have also identifi ed so-called "refugee proteins" that seem to be unrelated to known lipid droplet functions ( 4 ) that fall into several categories: membrane traffi cking proteins [Arf1, Rab, and Rho proteins ( 21 )], signaling proteins (PKC, Ras, AKAP), cytoskeletal proteins [actinin, fi lamin A, myosin heavy chain (21)], enzymes (alcohol dehydrogenase), chaperones [pyruvate kinase, peroxiredoxin 1 ( 21 )], and proteins associated with cellular organelles.
Membrane traffi cking proteins commonly observed in CLD preparations include proteins from several small GT-Pase families: Ras-related Rab proteins, ADP Ribosylation factor (ARF) proteins, as well as related proteins phospholipases and caveolins. Though the relationship between CLDs and the small GTPases is unclear, in vivo colocalization studies using immunofl uorescence seem to suggest that these interactions are not due to contamination during fractionation ( 44,65 ).
In particular, the Rab protein family of small Ras-related GTPase proteins is well represented in the proteomic studies of CLDs, whereas other members of the small GTPase families such as Rho and Arf proteins are less frequently observed. Rab proteins are known to be important for vesicular traffi cking, vesicle budding, and fusion. Although twenty Rab proteins were identifi ed in mammalian studies, Rab effector proteins were not observed in large numbers. The presence of both the small GTPase proteins and their effector proteins is needed for synthesis of a complete pathway. Effector proteins may simply not be present in CLDs or they could be present at such low abundances in CLDs prior to enrichment and purifi cation that they are either lost during enrichment or are below the limit of detection of the analysis. Another possibility is that the effector proteins are only recruited to CLDs under specifi c conditions ( 21 ). either A431 cells with low endogeneous stomatin levels (called stomatin-negative) or on A431 cells stably overexpressing wild-type stomatin (termed stomatin-positive cells). Comparing the 1D SDS-PAGE bands from stomatinpositive and stomatin-negative cells did not show differences in the protein bands observed through staining of the gels with the exception of the stomatin band. Proteins identifi ed included those previously associated with CLDs, including PAT family proteins, several Rab GTPases (Rab 1B, 6A, 7, 10, and 18), four lipid metabolism proteins, a fatty acid synthesis protein, four proteins involved in steroid synthesis or vitamin A homeostasis, four vesicle transport proteins, seven chaperones or chaperonins, three cytoskeletal proteins, and two uncategorized proteins.

SUMMARY OF PROTEOMIC STUDIES
Over two hundred mammalian proteins have been identifi ed across a number of proteomic analyses of enriched CLDs. [A comprehensive list of all identifi ed CLD proteins in yeast (110 proteins), Drosophila (296 proteins), and mammals (229 proteins) organized according to the function/cellular localization can be found in supplementary  Tables I , II, and III, respectively.] Table 1 summarizes the CLD proteins identifi ed in at least three proteomic studies from mammalian cells/tissues. These proteins seem to fall into several distinct functional categories.
The noted "gold standard" marker for CLDs are members of the PAT family [named for the fi rst three members perilipin, adipophilin (also known as adipocyte differentiation related protein, ADRP, or ADFP), and tail interacting protein 47 (TIP47), but the PAT-family also includes lipid storage droplet protein 5 (LSDP5, also known as OXPAT), myocardial lipid droplet protein (MLDP, also called PAT-1), and S3-12] ( 51 ). Though PAT proteins are all known to associate with CLDs, they are not all found in every cell. ADRP has been localized to lipid droplets in cultured murine 3T3-Ll adipocytes, murine MA-10 Leydig cells, CHO fi broblasts, and human HepG2 hepatoma cells ( 43 ) and TIP47 is similarly found in many cell types ( 52 ). However, perilipin has only been found in adipocytes and steroidogenic cells ( 53 ). The LSDP5 (OXPAT) protein is found in tissues that rely on ␤ -oxidation of fatty acids for energy production such as heart muscle, brown adipose tissue, and liver (54)(55)(56), and S3-12 is found in white adipose tissue ( 57 ). Two related proteins, LSD1 and LSD2, are expressed in insects and found in the fat body, a tissue that has similarities to both liver and adipose tissue (58)(59)(60)(61).
PAT proteins are thought to be responsible for lipid homeostasis within cells, but the distinctions and specifi cities of the family members are not yet fully understood. PAT proteins share a conserved 100 amino acid homologous N-terminal PAT domain (the exception is S3-12) ( 51 ) and repeating sequences of 11 amino acids thought to form amphipathic ␣ -helices that may assist in embedding the proteins within lipid droplets ( 33,34,51 ). Phosphoryla- model is that CLDs are formed by generation of lipid esters by the ER that are deposited into the membrane. As the quantity of lipid esters exceeds the molar proportion that can be assimilated within the phospholipid bilayer, the lipid ester begins to segregate between the two leafl ets. This process is represented in Fig. 1 . A budding process occurs whereby the lipid droplet begins to separate away from the membrane to form an independent lipid droplet in the cytoplasm ( 7 ). Micro-droplets of lipids are formed at the cisternae of the ER and aggregation of those microdroplets forms larger CLDs ( 10 ). Organelles other than the ER are also commonly found in close proximity to CLDs and CLDs have been found to contain proteins associated with the Golgi apparatus ( 73 ), peroxisomes ( 11,19 ), mitochondria ( 22 ), and the plasma membrane ( 24 ), and CLDs are frequently found in close physical contact with these organelles.

CONCLUSIONS AND PERSPECTIVES
The most interesting categories of proteins identifi ed through proteomic analysis are those that do not have known functions within the classical view of the CLD. These "refugee proteins" ( 4 ) have prompted an ongoing debate questioning whether these proteins are bona fi de CLD proteins. It has been suggested that CLDs may be more susceptible than other cellular structures or organelles to poor isolation due to the intrinsic hydrophobicity of the CLD structure ( 4,74 ). This potential for in vitro contamination is intrinsic to every fractionation protocol. Therefore, in vivo localization studies (immuno-electron Alternatively, the presence of incomplete and fragmented protein pathways on CLDs in vivo could be the result of CLDs acting as a reservoir of hydrophobic proteins within the cell that functions to transport and assist with synthesis of protein pathways ( 21 ). Transfer of substrates and products between CLDs and organelles or other cellular structures could be facilitated by the Rab proteins and other membrane traffi cking proteins.
The phospholipid monolayer surrounding CLDs contain abundant phosphatidylcholine and lysophosphatidylcholine ( 13 ) that are the substrates for phospholipase D (PLD) (reviewed in ref. 66 ). PLD1 is involved in many cellular processes like membrane fusion and vesicular traffi cking (reviewed in ref. 66 ). The small protein Arf1 directly activates PLD1, and has received particular attention with respect to its interactions with lipid droplets. Using immunofl uorescence, Arf1 and PLD1 have been shown to localize to CLDs ( 44 ) and Arf1 was shown to directly bind ADRP ( 65 ). A GDP-restricted form of Arf1 caused ADRP dissociation from CLDs and did not inhibit triacylglycerol formation but did prevent formation of new CLDs, which indicates a prevention of CLD budding ( 65 ). Arf1 was found in one proteomic study by Bartz et al. ( 21 ) along with the ADP-ribosylation factor GTPase-activating protein 1 (ArfGAP1, Uniprot: Q8N6T3).
Proteins associated with the ER are also commonly observed in CLD preparations. This is not surprising in light of the fact that lipid droplet biogenesis is thought to occur at the surface of the ER (reviewed in refs. 1, 2, 5, 7, 8, and 67 ). Numerous mechanisms for LD biosynthesis have been proposed (68)(69)(70)(71)(72). Currently, the most widely accepted needed ( 15 ). This mechanism has also been observed when the levels of protein expression are higher than normal due to unusual cellular metabolic conditions, as in the case of stomatin ( 24 ). Protein sequestration is a tantalizing idea because it could explain the many refugee proteins that have been found to associate with CLDs and provide a dynamic regulatory mechanism for their storage and transport ( 4 ). This idea fi ts appealingly with the known regulation of lipids that have been attributed to CLDs for many years. The CLD system could potentially act as a regulatory mechanism for both proteins and lipids within cells. Figure 1A illustrates the sequestration of binding partners, showing the sequestered and un-sequestered state.
Inactivation of proteins through sequestration to CLDs could happen either indirectly by separating proteins from binding partners, cofactors, or substrates, or directly through changing the binding of an enzyme by actively blocking a catalytic site or inducing a conformational change in the enzyme when it is bound to the CLD. Sequestration of proteins within CLDs may provide a buffering mechanism for proteins within cells, making proteins available as needed but keeping proteins separated from binding partners and preventing aggregation when proteins are not needed. Figure 1B illustrates the sequestration of binding partners, showing the sequestration on a CLD of an aggregation-prone protein, followed by release of the sequestered protein once an appropriate binding partner is available.
There are several features of CLDs that offer potential utility as a protein storage mechanism. First, the dynamic nature of CLDs' size and the number of CLDs can be adjusted based on external stimuli and metabolic state, allowing for a variable surface area and internal hydrophobic core. This ability would provide the capacity for CLDs to dynamically adjust when greater quantities of CLDs might be needed for storage of proteins. Second, the structure of CLDs with a hydrophobic core of neutral lipids surrounded by a phospholipid monolayer may provide an excellent microscopy and/or fl uorescence microscopy) are required for confi rmation of bona fi de CLD proteins.
Ideally, genetic studies would follow to validate functional signifi cance ( 58,61 ). Some of the CLD proteomic studies discussed above incorporated genetic knockout or knockdown strategies for functional validation in S. cerevisiae and Drosophila ( 16,18,19 ). A mouse variant knockout of perilipin has proven to be an extremely valuable tool for demonstrating that perilipin acts as a phosphorylationcontrolled gatekeeper that can regulate access to the neutral lipid core of the CLD for lipases, thereby acting as a control on the lipolysis of CLDs ( 45,46 ). Knockouts have been reported for adipose differentiation-related protein (also known as adipophilin, ADRP, or ADFP) ( 75 ) as have knockdown studies of ADRP using antisense oligonucleotides ( 76 ). Unfortunately, knockout or knockdown studies may sometimes produce ambiguous results that may not clearly defi ne a phenotype related to the reduction in a protein, and this case has been reported for the ADRP phenotype. [ ( 75,76 ) and reviewed in ( 63 )] Knockouts of other CLD-related proteins have been reported for the senescence marker protein-30 (SMP30) ( 77 ), adipocyte triglyceride lipase (ATGL) ( 78 ), apoA-II ( 79 ), hepatic lipases ( 79 ), ␣ -synuclein ( 80,81 ), hormone sensitive lipase ( 82,83 ), caveolin-1 ( 84 ), and vimentin ( 85 ). Knockouts of CLD-related proteins for other species have also been reported, such as the Drosophila adipose gene ( adp ) ( 86 ), and the Drosophila PAT protein analog LSD2 ( 58,61 ).

Sequestration of proteins on CLDs
One emerging idea is that CLDs may serve in a broad cellular role as a protein storage depot for sequestering proteins ( 15 ). In this capacity, CLDs would protect proteins from degradation while keeping aggregation-prone proteins physically separated from the cytoplasm. There is evidence in Drosophila that maternal histones, found in high abundance in embryos, are sequestered on the surface of CLDs and transferred rapidly on and off as they are Fig. 1. Proposed cellular functions of CLDs. CLD formation begins in the smooth endoplasmic reticulum. Lipid esters are deposited within the phospholipid bilayer and microlipid droplets form, bud, and are released into the cytoplasm ( 7 ) associated with PAT proteins (pink circles). Three potential CLD functional roles are: A, Sequestration: proteins may be reversibly inactivated by CLD sequestration of one binding partner. Active state and sequestered states are shown. B, Chaperone: aggregation prone proteins without binding partners are sequestered until appropriate binding partners are available. C, Protein transport: CLDs move along cytoskeletal components carrying sequestered proteins from one cellular location to another. storage site for proteins that have exposed hydrophobic surfaces. Third, the complement of proteins associated with CLDs can respond dynamically to physiological changes around the cells ( 19,22 ). There is some evidence that proteins may localize to specifi c CLDs within a single cell ( 16,(87)(88)(89)(90). Selective sequestration of proteins could potentially act to distribute proteins throughout cells using CLDs as transport vehicles, but evidence for this is limited at present. Figure 1C shows the hypothesized transport function of CLDs, with the CLD temporarily sequestering a protein for transport, and motion of CLDs along a cytoskeletal component (here, a microtubule).
The current CLD literature is growing, and proteomics has contributed several enticing functional leads. However, much remains to be done and the systematic validation of identifi ed CLD proteins promises to elucidate the full range of functions of CLDs within cells.