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273 New HIV Host Proteins Discovery Reported
HIV Gets By With a Lot of Help From Human Host
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FULL TEXT OF ORIGINAL SCIENCE FOLLOWS BELOW
Science 11 January 2008:
Vol. 319. no. 5860, pp. 143 - 144
Jon Cohen
HIV is ridiculously simple yet astonishingly complex. The virus contains a mere 9000 bases of RNA--one-millionth the amount of genetic material in a human cell--and a paltry suite of nine genes that code for a measly 15 proteins. Yet this virus can relentlessly nibble at immune cells until the entire system collapses, opening the door for a vast array of illnesses and, ultimately, death. For HIV to do its damage, however, it must repeatedly infect new cells and copy itself, a feat that requires help from its human host. And as a startling paper published online (www.sciencemag.org/cgi/content/abstract/1152725) by Science this week explains, that's where HIV's complexity becomes abundantly apparent. The findings also spotlight intriguing, novel drug targets. "This is destined to be one of the key HIV papers of this decade, if not longer," says Robert Gallo, who heads the Institute of Human Virology in Baltimore, Maryland, and did landmark studies that tied HIV to AIDS.
Using cutting-edge molecular techniques, a team led by geneticist Stephen Elledge at Brigham and Women's Hospital in Boston found that the virus relies on 273 human proteins to do its dirty work. These so-called HIV dependency factors (HDFs)--only 36 of which researchers had previously identified--enable the virus to attach to immune cells, wiggle in, shed the protein coat that surrounds its RNA, convert that to DNA, shuttle the genetic material into the nucleus, transcribe genes into amino acids, and then assemble proteins, sprinkle them with sugars, and help newly minted HIVs bud through the surface, where they then go on to find their own cellular prey. "Some viruses carry their houses on their backs, and other viruses invade other people's houses and take over," says Elledge, who had never done an HIV study before but was attracted by the virus's small size. "HIV is more of the latter, and it requires lots and lots of different host functions."
Elledge, postdoc Abraham Brass, and co-workers--including Judy Lieberman, director of the Division of AIDS at Harvard Medical School in Boston--found these HDFs by using libraries of recently discovered small interfering RNAs (siRNAs), which can disrupt transcription and thereby prevent genes from making their products. Specifically, they took human cells and effectively short-circuited every known gene, one at a time, and then tested whether HIV could establish an infection and copy itself. In all, their genomewide RNA interference screen disrupted more than 21,000 human genes, and by a process of elimination, they isolated the ones that HIV hijacks. "This is an excellent example of siRNA screening," says retrovirologist Warner Greene, who heads the Gladstone Institute of Virology and Immunology at the University of California, San Francisco. "This single paper could guide several interesting graduate student theses in the future."
Gallo says the findings have already led to many new insights, and he shares the study investigators' enthusiasm that these HDFs may make excellent targets for drugs. Elledge compares the strategy to that of much-ballyhooed cancer drugs known as angiogenesis inhibitors, which strangle the blood supply to tumors rather than attack the tumors themselves.
More than two dozen current drugs disable key HIV enzymes. (The U.S. Food and Drug Administration in August for the first time approved an HDF inhibitor, which blocks a receptor the virus docks onto for cell entry called CCR5, but its use is limited to people who have failed to respond to several other drugs.) Although drugs that cripple HIV work powerfully when combined into cocktails, the virus can mutate around each of them, preventing them from binding to their viral targets, eventually leading to drug resistance. Elledge and co-workers contend that HIV would have more difficulty escaping drugs that interfere with HDFs. True, HIV could evolve the capacity to copy itself without one of these factors, but that's a much more difficult task for the virus than mutating to prevent a drug from binding to a viral enzyme. On the flip side, human proteins don't mutate with anywhere near the ease of viruses, which makes it less likely that an HDF would develop drug resistance.
Greene and others caution that targeting host proteins could lead to serious side effects--after all, these HDFs presumably exist to help humans, not the virus. It's also a tall order to discover effective inhibitors against HDFs, says Deborah Nguyen, who with colleagues at the Genomics Institute of the Novartis Research Foundation in San Diego, California, recently published a more limited siRNA study to identify new HIV treatment strategies. "Unfortunately, I think this barrier won't be crossed for a while," predicts Nguyen, who says industry's interest in anti-HIV drug R&D is also waning.
Elledge acknowledges the hurdles but counters that many marketed drugs against other diseases target human proteins and provide more benefit than harm. And the hundreds of HDFs his group has identified may play limited roles in human health and development. "Perturbing one may not have a profound effect on a cell, but it may on HIV," he says. Yet he agrees that this flood of new data is confusing: "It takes some hard thinking about where to go next."
Greene says the most immediate challenge is to elucidate the molecular details of how these 273 HDFs interact with HIV. "Currently, the authors can only suggest possible connections," he says. "But what a great starting point."
Identification of Host Proteins Required for HIV Infection Through a Functional Genomic Screen
Abraham L. Brass,1,2 Derek M. Dykxhoorn,3* Yair Benita,4* Nan Yan,3 Alan Engelman,5 Ramnik J. Xavier,2,4 Judy Lieberman,3 Stephen J. Elledge1
1Department of Genetics, Center for Genetics and Genomics, Brigham and Women's Hospital, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA. 2Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA. 3Immune Disease Institute and Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA. 4Center for Computational and Integrative Biology, Harvard Medical School, Boston, MA 02114, USA. 5Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, MA 02115, USA.
These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail:
selledge@genetics.med.harvard.edu
HIV-1 exploits multiple host proteins during infection. We performed a large-scale siRNA screen to identify host factors required by HIV-1 and identified over 250 HIV-dependency factors (HDFs). These proteins participate in a broad array of cellular functions and implicate new pathways in the viral lifecycle. Further analysis revealed previously unknown roles for retrograde Golgi transport proteins (Rab6 and Vps53) in viral entry, a karyopherin (TNPO3) in viral integration, and the Mediator complex (Med28) in viral transcription. Transcriptional analysis revealed that HDF genes were enriched for high expression in immune cells suggesting that viruses evolve in host cells that optimally perform the functions required for their lifecycle. This effort illustrates the power with
which RNA interference and forward genetics can be used to expose the dependencies of human pathogens such as HIV, and in so doing identify potential targets for therapy.
Discussion. Judging from the diverse cellular processes detected in our screen, the exploitation of host cell functions by HIV is extensive. The functional clustering and confirmation of HDFs provides internal validation for our screen and suggest that the majority of the 200 proteins identified with no previous links to HIV are likely to play relevant roles in HIV pathogenesis. We have portrayed the HIV viral lifecycle along with the presumed subcellular locations and functions of the novel and known HDFs in Fig. 5 (rationale provided in table S4 (4)).
Additional validation comes from the analysis of the enrichment of genes connected to known proteins implicated in HIV function. We find a strong enrichment for connectivity to this dataset (fig. S7, table S5 (4)). Furthermore, although the screen was performed in HeLa cells, HDFs were significantly enriched for high expression in immune cells. This suggests that immune cells are especially proficient for the functions HIV needs for optimal replication, and became the selected host for that reason. It will be interesting to determine if the virus is especially reliant on this set of proteins and whether the tropism of other viruses can be similarly explained.
Rab6 and Vps53 are required for viral fusion to the membrane through an unknown mechanism. While we have ruled out alteration of host co-receptor cell surface expression, several alternative explanations exist. There could exist a previously undetected co-receptor, dependent on Rab6 and Vps53. The screen identified over 30 transmembrane proteins with no known HIV association (table S2).
Modification of host receptors may be aberrant. However, no host receptor modification is known to be required for infection (27, 28). Alternatively, the plasma membrane composition may be affected, possibly due to alterations in the major supplier of membrane, the Golgi. Among the candidates affected by such a perturbation are the glycosphingolipids (GSLs), which are required for HIV-host cell fusion (29-31). GSLs are synthesized by ER and Golgi enzymes (32) that depend on retrograde vesicular transport for recycling (18, 33). Rab6/Ypt6 mutants are defective in retrograde Golgi transport, resulting in vesicular scattering and lysosomal degradation of many Golgi resident enzymes, possibly altering GSL homeostasis (18, 20) (supporting online text).
The HIV PIC accesses our genome through the NPC. A candidate for the PIC-associated karyopherin is TNPO3, whose depletion profoundly blocked provirus formation. This effect might be indirect if TPNO3 is required for the activity of another HDF. However, a simpler model involves the PIC binding to TNPO3, then entering the nucleus via interactions with two NPCs detected in the screen, RanBP2 and Nup153 (34). While speculative, these are examples of the kinds of detailed hypotheses that can be generated from a highly validated, functionally-derived dataset.
A key pharmacologic strategy for treating individuals living with HIV has been to simultaneously target multiple virus-encoded enzymes required for replication to overcome emergence of drug resistance. We have taken a parallel strategy by identifying host factors required for the HIV life cycle. Such proteins represent therapeutic targets that are not plagued by the twin problems of viral diversity and escape mutation that interfere with the effectiveness of conventional anti-retroviral drugs. We anticipate that HIV would be hard-pressed to evolve resistance to drugs targeting cellular proteins, because it would have to evolve a new capability, not simply mutate a drug-binding site. This is analogous to blocking angiogenesis in non-tumor cells to deprive cancer of a blood supply (35). Support for the notion that these HDFs may represent potential therapeutic targets, arises in part from a recent genome association study reporting that single nucleotide polymorphisms in ZNRD1 are associated with slowed HIV disease progression (36). Our screen found ZNRD1 depletion inhibited HIV (table S2). This suggests that variants in other HDFs might modulate HIV infection and drugs inhibiting their functions may prove protective.
HIV-1 encodes only fifteen proteins (1) and thus must exploit multiple host cell functions for successful infection (2). Viral entry depends on binding to the receptor CD4 and either of two co-receptors, CXCR4 or CCR5. Upon membrane fusion, the viral core, containing the viral capsid and nucleocapsid along with the viral genome, reverse transcriptase (RT), integrase (IN), protease (PR) and the viral accessory proteins Vif, Nef and Vpr, is released into the cytoplasm. Collectively called the reverse transcription complex (RTC), this assembly binds to actin, triggering the synthesis of a double stranded viral DNA complement forming the preintegration complex (PIC), which moves along microtubules to the nucleus and enters via a nuclear pore.
IN binds to host LEDGF, and catalyzes HIV DNA integration. Proviral transcription depends on the viral factor, Tat, which binds to the transactivation response element (TAR) in the proviral RNA and promotes elongation by recruiting Cyclin T1, HTATSF1, and Cdk9. Unspliced and partially spliced HIV transcripts require the viral Rev protein for nuclear export. HIV buds directly from the plasma membrane through association with the host Class E Vps proteins (3). Because of the complexity of the retroviral life cycle and the small number of viral proteins, important viral-host relationships likely remain to be discovered. Toward this goal, we performed a genome-wide RNAi screen to identify host factors involved in HIV-1 infection.
The siRNA screen. We developed a two-part screen to detect host proteins needed for HIV infection (Fig. 1A) (4). Part one consisted of infecting siRNA transfected cells with the IIIB strain of HIV-1 (HIV-IIIB, (4)), supplemental online text) and 48 hours later staining for p24, produced from the HIV gag gene. This detects host proteins needed from viral entry through Gag translation, but is less sensitive for factors affecting viral assembly and budding. To identify late-acting
factors, we performed part two by incubating culture supernatants from part one with fresh reporter cells and assaying for Tat-dependent reporter gene expression after 24 h. For the screen, we employed HeLa-derived TZM-bl cells, which express endogenous CXCR4, transgenic CD4 and CCR5, and an integrated Tat-dependent beta-galactosidase reporter gene. The screen was optimized using siRNAs against Tat, CD4 and Rab9p40, the latter required for HIV particle release (5). siRNAs targeting CD4 or Tat, produced >3-fold decrease in p24 expression (Fig. 1B and C). Only modest, protection was seen upon Rab9p40 depletion (Fig. 1C, part one). However, after incubation of fresh TZM-bl cells with transferred cultured supernatant, the depletion of Rab9p40 scored convincingly (Fig. 1C, part two).
This platform was used for a genome wide screen. The siRNA library contains 21,121 pools of four siRNAs per gene. Pools were classified as hits if they decreased the percentage of p24 positive cells or beta_galactosidase activity by two or greater standard deviations (SD) from the plate mean (table S2, Column E (4)). We also required that the siRNAs did not decrease viable cells by greater than two SDs. These criteria were met by 386 pools (1.8%). We next rescreened the four siRNAs from each pool separately. In this validation screen, 273 pools (71%) reconfirmed with at least one siRNA scoring in either part one or two. There was a strong correlation between parts one and two. Only 28 genes appeared specifically in part two, suggesting these factors act in late stages of infection (table S2, supplemental online text).
HDF bioinformatics analysis. Of the confirmed HDFs, we identified 36 host factors (13%) previously implicated in HIV pathogenesis (table S1), including CD4, CXCR4, NMT1, Rab9p40, and components of the NF-_B and CREB
transactivation pathways. Among the 237 remaining genes, over 100 had two or more individual siRNAs score, reducing the likelihood of off-target effects (table S2). Subcellular localization, gene ontology (GO) biological processes and
molecular functions of the candidates are shown (Fig. 1D, E, fig. S1A, B, and table S3). 136 GO biological process terms, assigned to 103 genes, were significantly enriched. Analysis of GO molecular functions identified enrichment for 17 nonredundant statistically significant terms assigned to 86 genes.
Several macromolecular complexes were also detected. The nuclear pore (NPC) Nup160 subcomplex, had 4 of 6 subunits identified (Nup85, Nup107, Nup133, Nup160). Since Nup160 complexes are NPC scaffolds, their loss may impede HIV nuclear access (6). Depletion of components of Mediator (Med4, Med6, Med7, Med14, and Med28), which directly couples transcription factors to RNA pol II (7), inhibited infection, perhaps shedding light on the requirements for activators that bind the viral long terminal repeat (LTR). Two ER-Golgi-associated assemblages, the conserved oligomeric Golgi (COG) complex (8) and the transport protein particle (TRAPP) I complex (9), also scored with multiple components, perhaps due to HIV's dependency on transmembrane glycoproteins for entry. Three of the late acting HDFs found in part two, OST48, STT3A and DPM1, encode enzymes involved in glycosylation (10, 11). HIV Env requires glycosylation to be infectious (12). Early studies showed glycosylation inhibitors prevent Env modification and blocked HIV fusion (13, 14).
Among the unexpected associations was autophagy, a process essential for the degradation and recycling of cellular components. Targeted substrates are encapsulated in membrane bound autophagosomes by two protein conjugation pathways (15). HIV infection depended on the presence of members of both pathways (Atg7, Atg8, Atg12, and Atg16L2). In addition, lysosomal-associated HDFs (CLN3, and LapTM5) may also be required for autophagy. HeLa cells are not the natural host for HIV. We wondered whether HDFs showed an expression bias in other cell types that might explain HIV tropism. We assessed the expression of 239 genes that were expressed in at least one of the 79 tissues in the Genomic Institute of the Novartis Research Fund (GNF) dataset, and found that 79/239 (33%) were enriched for high expression in immune cells (p<0.001, top 7% expression, Fig. 1F, table S3 (4)).
HIV entry requires retrograde vesicular transport.
Rab6A, regulates retrograde Golgi-to-ER transport, and is important for recycling of Golgi-resident enzymes (16, 17). Three of four siRNAs confirmed in the validation round for both Rab6A and Rab6A_, which differ by three amino acids
due to alternative splicing (table S2) (16). Rab6A_ controls endosomal trafficking, and is the homolog of the yeast Ypt6 (16); both isoforms will hence be referred to as Rab6. Ypt6 mutants are defective in retrograde Golgi transport, particularly recycling of glycosyltransferases (17, 18). Depletion of the homolog of Rgp1p, a guanine nucleotide exchange factor required for Ypt6 function, also decreased
HIV infection (table S2 (19)).
We generated cells stably expressing short hairpin RNA (shRNAs) directed against Rab6. All three shRNAs decreased infection proportional to Rab6 depletion (Fig. 2A-C) in the first phase of the lifecycle since Rab6 depletion inhibited Tat-dependent reporter expression 20 hours post-infection (Fig. 2B). Expression of Rab6-GFP lacking the Rab6 3_UTR targeted by the shRNAs, rescued susceptibility to infection (Fig. 2A-C, fig. S2). Given Rab6's role in vesicular transport, we examined surface expression of CD4 and CXCR4 by
FACS in the Rab6 knockdown (Rab6-KD) lines. CXCR4 expression showed minor variations that did not correlate with resistance to HIV or Rab6 depletion and were not restored upon Rab6 repletion (fig. S3A, B). CD4 levels were unaltered (fig. S3C). Thus, something other than receptor expression is defective in Rab6-KD cells.
To characterize the block to infection further, we infected Rab6-KD cells with either HIV-IIIB, or an HIV strain pseudotyped with the vesicular stomatitis virus G envelope protein (VSV-G), containing a yellow fluorescent reporter in place of nef (HIV-YFP). Only HIV-IIIB infection was inhibited (Fig. 2D). In addition, VSV-G pseudotyped Moloney leukemia virus (MLV-EGFP) infection was unperturbed. HIV envelope proteins promote fusion of the virus to the cell membrane. In contrast, VSV-G pseudotypes are endocytosed, with endosomal acidification triggering fusion. Thus, our initial observations suggested Rab6 acted early in infection. HIV-IIIB has tropism for the co-receptor CXCR4. To determine whether inhibition was restricted to CXCR4 virus, we examined the effect of Rab6 silencing on infection with HIV-Bal, a CCR5 tropic virus. Targeting Rab6
did not alter surface CCR5 expression (fig. S3G), but did inhibit HIV-Bal infection (Fig. 2E). Therefore, Rab6 plays a role in infection by both CCR5 and CXCR4 viruses. Rab6 was required for late reverse transcription of the viral genome, indicating an early block (Fig. 2F). We therefore tested Rab6's role in fusion by co-culturing HL2/3 HeLa cells, which express Tat and HIV receptor proteins gp41 and gp120, with TZM-bl cells. HL2/3 viral receptors interact with CD4 and CXCR4 on TZM-bl cells, prompting cellular fusion via the same mechanism used by HIV. Upon fusion, Tat activates beta-galactosidase expression. Decreased Rab6 levels correlated with diminished beta-galactosidase activity, consistent with inhibition of viral fusion (Fig. 2G, fig. S4C (4)). To test Rab6's role in HIV infection in a more physiologic cell, we transfected a T cell line, Jurkat, with Rab6 siRNAs, then infected with HIV. Reduced infection was seen after transfection with two of three Rab6 siRNAs tested (Fig. 2H) correlating with Rab6 depletion (Fig. 2I). No effect on receptor levels was observed in the transfected T cells (fig.
S3E, F).
Similar results were obtained for Vps53 (figs. S4A-F, S5). Yeast Vps53 is a component of the Golgi associated retrograde protein (GARP) complex. GARP tethers transport vesicles trafficking from endosomes to the trans-Golgi network (TGN) in an Ypt6-dependent manner (20-23). Together, these data suggest retrograde trafficking of vesicles from endosomes to the Golgi is needed for HIV infection, possibly at viral entry.
A role for a karyopherin in HIV replication.
Transportin 3 (TNPO3), a karyopherin, imports multiple proteins into the nucleus, including histone mRNA stem-loop binding protein (SLBP, (24)), serine/arginine-rich proteins (SR proteins) that regulate splicing of mRNA (25) and repressor of splicing factor (RSF1, (26)). Eight of eight TNPO3 siRNAs lowered infection in HeLa cells (Fig. 3A, H). TNPO3 mRNA reduction, as determined by quantitative PCR (qPCR), correlated with the inhibition of infection (Fig. 3E). Prevention of infection by TNPO3 silencing was independent of HIV envelope (Fig. 3B). TNPO3 depletion also inhibited infection of Jurkat cells (Fig. 3D).
TNPO3 depletion did not affect MLV-EGFP (Fig. 3C); This could be explained if TNPO3 depletion impaired SR protein-dependent splicing of Tat, which is required for efficient HIV, but not _-retroviral, transcription. However, Tat-dependent reporter gene expression from a transiently transfected HIV-YFP plasmid was only weakly affected by TNPO3 depletion (Fig. 3B). Additionally, an HIV derivative, pHAGE-CMV-ZSG, that contains HIV Gag and Pol, but expresses a fluorescent reporter protein from an internal CMV promoter, also showed a dependency on TNPO3 upon viral infection, but not plasmid transfection (Fig. 3C). These observations suggest that TNPO3 is needed prior to viral mRNA splicing. Assays for late RT products and integrated viral DNA in TNPO3-depleted cells showed that the block occurred after reverse transcription but prior to integration (Fig. 3F, G). Thus, diminished TNPO3 produces a lentiviral specific pre-integration block, perhaps at the stage of PIC nuclear import. Whether TNPO3 directly interacts with the virus or indirectly, via altered import or splicing of an HDF required for integration, remains to be determined.
The Mediator complex in HIV infection. Depletion of several components of Mediator inhibited HIV infection. We focused on Med28 because all four Med28 siRNAs inhibited first round HIV infection (Fig. 4A, fig. S6). Med28 depletion also protected Jurkat cells and efficiently decreased target gene protein levels (Fig. 4C and D). Loss of Med28 appeared to specifically affect HIV because it inhibited both HIV-IIIB and HIV-YFP, but not MLV (Fig. 4B). We found no decreases in reverse transcribed cDNA or integrated proviral DNA upon Med28 depletion (Fig. 4E and F). However, Med28 loss also decreased YFP expression from a transiently transfected HIV-YFP plasmid (Fig. 4G). Therefore, we conclude that Med28 is required for transcription of viral genes, consistent with its connection to RNA Pol II.
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