The Role of LIP5 and CHMP5 in Multivesicular Body Formation and HIV-1 Budding in Mammalian Cells

Abstract

We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release. We examined the function of LIP5 in mammalian cells, because the yeast homologue Vta1p was recently identified as a protein required for multivesicular body (MVB) formation. LIP5 is predominantly a cytosolic protein. Depletion of LIP5 by small inhibitory RNA (siRNA) does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does reduce the degradation of internalized epidermal growth factor receptor (EGFR), with EGFR accumulating in intracellular vesicles. Depletion of LIP5 by siRNA also decreases human immunodeficiency virus type 1 (HIV-1) budding by 70%. We identify CHMP5 as a LIP5-binding protein and show that CHMP5 is primarily cytosolic. Depletion of CHMP5 by siRNA does not affect the distribution or morphology of early endosomes, lysosomes, or Golgi but does result in reduced degradation of the EGFR similar to silencing of LIP5. Surprisingly, CHMP5 depletion results in an increase in the release of infectious HIV-1 particles. Overexpression of CHMP5 with a large carboxyl-terminal epitope affects the distribution of both early and late endocytic compartments, whereas overexpression of LIP5 does not alter the endocytic pathway. Comparison of overexpression and siRNA phenotypes suggests that the roles of these proteins in MVB formation may be more specifically addressed using RNA interference and that both LIP5 and CHMP5 function in MVB sorting, whereas only LIP5 is required for HIV release. Endosomes play a crucial role in transporting molecules from the plasma membrane to intracellular compartments as well as transporting molecules from the biosynthetic apparatus to their site of action (1Gruenberg J. Maxfield F.R. Curr. Opin. Cell Biol. 1995; 7: 552-563Crossref PubMed Scopus (554) Google Scholar, 2Clague M.J. Biochem. J. 1998; 336: 271-282Crossref PubMed Scopus (147) Google Scholar, 3Bishop N.E. Int. Rev. Cytol. 2003; 232: 1-57Crossref PubMed Scopus (41) Google Scholar). Several different endosomal compartments have been described based on morphology, constituents, and role within the endocytic apparatus. The multivesicular body (MVB) 1The abbreviations used are: MVB, multivesicular body; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GST, glutathione S-transferase; HIV-1, human immunodeficiency virus type 1; PBS, phosphate-buffered saline; siRNA, small inhibitory RNA; VPS, vacuolar protein sorting; GFP, green fluorescent protein; HPLC, high pressure liquid chromatography; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; Tf, transferrin. is an endosomal compartment that serves to sort membrane proteins destined for degradation or routing to the lysosome (4Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar, 5Piper R.C. Luzio J.P. Traffic. 2001; 2: 612-621Crossref PubMed Scopus (164) Google Scholar, 6Raiborg C. Rusten T.E. Stenmark H. Curr. Opin. Cell Biol. 2003; 15: 446-455Crossref PubMed Scopus (408) Google Scholar). These proteins are internalized into vesicles that form by the invagination of the limiting membrane (forming intravesicular vesicles). The contents of the MVB are then transferred to lysosomes. The physiologic importance of MVBs has been shown through studies in organisms as diverse as yeast and humans (5Piper R.C. Luzio J.P. Traffic. 2001; 2: 612-621Crossref PubMed Scopus (164) Google Scholar, 7Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar, 8Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (436) Google Scholar, 9Bishop N. Woodman P. Mol. Biol. Cell. 2000; 11: 227-239Crossref PubMed Scopus (214) Google Scholar, 10Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 11Morita E. Sundquist W.I. Annu. Rev. Cell Dev. Biol. 2004; PubMed Google Scholar). In yeast, the role of the MVB has been defined through the identification of mutations that alter protein sorting through the MVB (12Raymond C.K. Howald-Stevenson I. Vater C.A. Stevens T.H. Mol. Biol. Cell. 1992; 3: 1389-1402Crossref PubMed Scopus (680) Google Scholar). A specific class of sorting mutants, class E vacuolar protein sorting (VPS) mutations, results in the accumulation of transport cargo in a prevacuolar compartment and a selective deficit in the delivery of that cargo to vacuoles (7Katzmann D.J. Babst M. Emr S.D. Cell. 2001; 106: 145-155Abstract Full Text Full Text PDF PubMed Scopus (1130) Google Scholar, 13Odorizzi G. Babst M. Emr S.D. Cell. 1998; 95: 847-858Abstract Full Text Full Text PDF PubMed Scopus (554) Google Scholar, 14Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (280) Google Scholar, 15Babst M. Katzmann D.J. Estepa-Sabal E.J. Meerloo T. Emr S.D. Dev. Cell. 2002; 3: 271-282Abstract Full Text Full Text PDF PubMed Scopus (693) Google Scholar, 16Babst M. Katzmann D.J. Snyder W.B. Wendland B. Emr S.D. Dev. Cell. 2002; 3: 283-289Abstract Full Text Full Text PDF PubMed Scopus (526) Google Scholar). The class E phenotype is described as an enlarged late endosomal compartment, which presumably arises because of an inability to invaginate the limiting membrane that would normally form the MVB. In higher eukaryotes, studies on the internalization and degradation of growth factor receptors as well as studies on viral budding have helped to elucidate the importance of MVBs, because many enveloped RNA viruses utilize components of the MVB to bud from cells (8Futter C.E. Pearse A. Hewlett L.J. Hopkins C.R. J. Cell Biol. 1996; 132: 1011-1023Crossref PubMed Scopus (436) Google Scholar, 11Morita E. Sundquist W.I. Annu. Rev. Cell Dev. Biol. 2004; PubMed Google Scholar). Recently, Vta1p was shown to play a role in MVB sorting in Saccharomyces cerevisiae, and it was shown that Vta1p interacts with Vps4p (17Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar, 18Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Analysis of mammalian Vps4p/SKD1-interacting proteins identified a protein, SBP1 (19Fujita H. Umezuki Y. Imamura K. Ishikawa D. Uchimura S. Nara A. Yoshimori T. Hayashizaki Y. Kawai J. Ishidoh K. Tanaka Y. Himeno M. J. Cell Sci. 2004; 117: 2997-3009Crossref PubMed Scopus (41) Google Scholar). Examination of genomic data bases showed that SBP1 (or the data base gene name DRG-1) was homologous to Vta1p, with most of the homology in the carboxyl terminus of the reported sequence. A yeast two-hybrid screen identified LIP5 as a protein that interacted with CHS1/Lyst (20Tchernev V.T. Mansfield T.A. Giot L. Kumar A.M. Nandabalan K. Li Y. Mishra V.S. Detter J.C. Rothberg J.M. Wallace M.R. Southwick F.S. Kingsmore S.F. Mol. Med. 2002; 8: 56-64Crossref PubMed Google Scholar). LIP5 and DRG-1 are identical with the exception that the carboxyl terminus of LIP5 is distinct from that of DRG-1. Our sequencing of LIP5 cDNA is in accord with the suggestion of Fujita et al. (19Fujita H. Umezuki Y. Imamura K. Ishikawa D. Uchimura S. Nara A. Yoshimori T. Hayashizaki Y. Kawai J. Ishidoh K. Tanaka Y. Himeno M. J. Cell Sci. 2004; 117: 2997-3009Crossref PubMed Scopus (41) Google Scholar) that LIP5 is, in fact, the same as DRG-1. The sequence of LIP5/DRG-1 is homologous to Vta1p; the two proteins show 20.2% identity and 48.9% similarity. In this paper we demonstrate that LIP5 is homologous to Vta1p and like Vta1p functions in MVB sorting. Depletion of LIP5 by siRNA reduces the degradation of epidermal growth factor receptor (EGFR) and reduces HIV-1 budding. We further show that LIP5 specifically interacts with CHMP5/Hspc177, the homologue of the S. cerevisiae class E protein Vps60p (21Kranz A. A. R. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). Depletion of CHMP5 by RNA interference the degradation of EGFR, but in to CHMP5 silencing to an increase in HIV release. we show that are on the of the endocytic apparatus in cells for CHMP5 with cells in which CHMP5 with a large epitope is and cells, cells, and cells in with and epidermal growth factor from and CHMP5 from and M. B. Ward D.M. E. T. D.M. A. Kaplan J. Sundquist W.I. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) using in I. of LIP5 cDNA that the sequence was We that the sequence is because of a in an early The cDNA a protein of into or was from the same and into and from and into and as described M. B. Ward D.M. E. T. D.M. A. Kaplan J. Sundquist W.I. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). used in are in I. of the the of and RNA used in this RNA of in a was into and as a protein in as the LIP5 protein was on CHMP5 was into in E. as a protein and the as the LIP5 or CHMP5 proteins with and into with protein, was and for by and and type cells and to for to The cells with using to the was by cells in with and and the by and cells using a A was from an and membrane and cytosolic by for The in and protein to for protein using a and of The proteins transferred to and with a of or by in a of and used a of by a The using cells, cells, or or proteins using an with a The cells or proteins using with PBS, or The cells in with or and for or EGFR or receptor a from of was used a of The and in with and The cells using an with a The using the on an with a using cells and into with and and for The cells using with The and was to of and The and with The with and then with the on transferred to and for proteins of Analysis of the on and by of and by the of protein was transferred to a and was then by a and in and in a Analysis was using a to a with was in of and was an A of A was was was for of The in play for of and The for was of was with for protein RNA of LIP5 and CHMP5 cDNA and a sequence from RNA are in I. siRNA for was as described J.E. M. Sundquist W.I. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). on cells or using with siRNA a of the cells and the cells for or in PBS, with by and for or cells in with of siRNA using as described J.E. M. Sundquist W.I. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). The cells with an HIV-1 the of L. D. Sci. S. A. 1996; PubMed Scopus Google of T. R. M. M. D. L. J. 1998; PubMed Google of R.C. Sci. S. A. 1996; PubMed Scopus Google and of the the and the cells the same for virus in and and was by human cells with the in the cells with with PBS, and with an and the from from and from and of the role of LIP5 in membrane in mammalian cells, we a a LIP5. The of the LIP5 was examined by and The LIP5 protein, and this be by of with LIP5 LIP5 has a of but the protein showed an of on The yeast LIP5 Vta1p, also more by sequence S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). the of LIP5 in cells a membrane was and both membrane and cytosolic examined by and The LIP5 two a membrane protein of and a protein of which we the with LIP5. the was not the was with we that the is a protein by the the was to LIP5 by it was to LIP5 by or LIP5 we the LIP5 cDNA into with an or a carboxyl-terminal The in cells or Examination by and showed that or not was cytosolic. has a of but showed a higher by Overexpression of or not affect membrane in the endocytic or as by of specific results for receptor and EGFR not The distribution of a which is in and lysosomes, was overexpression of of a role for LIP5 in MVB of LIP5 was by cells with siRNA LIP5. of LIP5 protein was in cells with a siRNA or siRNA not The in as by was The cells with siRNA LIP5 showed in the distribution of or The yeast homologue of Vta1p, a role in MVB and of results in a in the degradation of internalized plasma membrane proteins (17Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar, 18Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). the of LIP5 affects the degradation of plasma membrane proteins in mammalian cells, we examined the of LIP5 silencing on the internalization and degradation of In cells, cells or cells with a siRNA the of in the degradation of EGFR In the of to LIP5 cells to degradation with a in EGFR The of RNA interference on the of EGFR A in EGFR degradation a in or a in internalization of receptor from the these we examined cells for EGFR distribution by EGFR is predominantly on the The of to cells or cells with a siRNA in the of EGFR and a in the In cells of the of in the of EGFR from the and the accumulation of EGFR in large intracellular vesicles. The accumulation of EGFR was in of the cells for LIP5. with not affect receptor or distribution in LIP5 cells not These results demonstrate that LIP5 is in the sorting and of the In the of the degradation of EGFR is and EGFR in intracellular vesicles. LIP5 with LIP5 interacts with we in cells, cells with and LIP5 using by with of the two and a protein of which was identified by as protein to a of small proteins (21Kranz A. A. R. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). The S. cerevisiae homologue of CHMP5 is a class E protein role in MVB formation has not been defined (4Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar). We a to The two in but a was the was with CHMP5 on these data and silencing data we that the protein is by showed CHMP5 to be primarily cytosolic The CHMP5 not for or CHMP5 in we a CHMP5 protein with a carboxyl-terminal or or was on vesicles the of and in a cytosolic protein, with the vesicles. are mammalian proteins that a of a is that proteins with LIP5 through sequence or this we and CHMP5 the into cells, the proteins using and examined the for LIP5. of in the of LIP5 of or not result in the of LIP5. These data demonstrate that the of LIP5 and CHMP5 is CHMP5 in the have shown that of proteins with a large or carboxyl-terminal epitope the endocytic apparatus M. B. Ward D.M. E. T. D.M. A. Kaplan J. Sundquist W.I. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C.R. J. Cell Sci. 2001; PubMed Google Scholar, J. A. D. A. Sci. S. A. 2003; PubMed Scopus Google Scholar, B. A. S. E. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C. H. S. K. M. K. T. Y. M. Biochem. J. 2004; Scholar). of not or the distribution of early as well as late endocytic and EGFR In overexpression not alter early or late endocytic Overexpression of or in an of not or was to be with In as by or CHMP5 with a carboxyl-terminal epitope was cytosolic not The or are to as M. B. Ward D.M. E. T. D.M. A. Kaplan J. Sundquist W.I. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C.R. J. Cell Sci. 2001; PubMed Google Scholar, J. A. D. A. Sci. S. A. 2003; PubMed Scopus Google Scholar). the role of CHMP5 in endocytic more we the of overexpression to depletion of CHMP5 by with siRNA specific for CHMP5 showed an in CHMP5 protein as by in CHMP5 protein was in cells with or Depletion of CHMP5 by siRNA not alter the distribution of or CHMP5 depletion affect EGFR CHMP5 protein degradation of the EGFR was with a of with in cells with and depletion of both CHMP5 and LIP5 not to further in the of degradation of EGFR, and the morphology of the vesicles in which EGFR was similar to LIP5 silencing not the of to cells, the EGFR was in enlarged vesicles with These vesicles the EGFR vesicles in cells that of that overexpression of a large CHMP5 to a different phenotype with CHMP5 A small but in EGFR was in cells of LIP5 with cells of EGFR to in enlarged vesicles in cells of whereas in cells of CHMP5 EGFR in that showed a distribution in LIP5 HIV to the sorting and degradation of internalized membrane depletion or of a of MVB proteins in mammalian cells reduces HIV-1 budding E. Sundquist W.I. Annu. Rev. Cell Dev. Biol. 2004; PubMed Google Scholar, A. J. 2004; PubMed Scopus Google Scholar, 2004; 106: PubMed Scopus Google Scholar, A. J. Cell Biol. 2003; PubMed Scopus Google Scholar). LIP5 or CHMP5 also play a role in viral we the release of and infectious HIV-1 from cells with siRNA to or siRNA the sequence was also used as a J.E. M. Sundquist W.I. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). proteins as by of and the intracellular not depletion of of the depletion reduce the release of and the infectious of LIP5 depletion also reduced viral release and of the of LIP5 depletion of the in release and specific and and that LIP5 is required for viral release and Surprisingly, depletion of CHMP5 viral release and of the in different the by CHMP5 We that the of CHMP5 in the viral release and is the in which depletion of a protein in MVB formation viral release. The S. cerevisiae homologue of Vta1p, is a protein in MVB formation (17Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar, 18Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). cytosolic proteins are required for MVB sorting in S. cerevisiae, and many of these proteins are based on or the in which are (4Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar, G. Katzmann D.J. Babst M. A. Emr S.D. J. Cell Sci. 2003; 116: PubMed Scopus Google Scholar). These are and with the to the in which in MVB formation (4Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar). of the proteins required for MVB formation in yeast have mammalian (4Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar, 10Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (240) Google and studies show their in MVB formation N. Woodman P. Mol. Biol. Cell. 2000; 11: 227-239Crossref PubMed Scopus (214) Google Scholar, M. B. Ward D.M. E. T. D.M. A. Kaplan J. Sundquist W.I. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J.E. M. Sundquist W.I. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. N. I. C. J. S. J. R. J. 2004; PubMed Scopus Google Scholar, A. A. Stenmark H. J. Cell Biol. 2003; PubMed Scopus Google Scholar, N. Woodman P. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). we two of that the mammalian of Vta1p, a role in mammalian MVB formation. EGFR is a MVB sorting pathway. We demonstrate that the of LIP5 by siRNA decreases the of degradation of EGFR and that EGFR in a mammalian class E We also demonstrate that the in EGFR degradation in LIP5 cells is not to in EGFR or in LIP5 also reduces the of infectious HIV-1 particles. mammalian proteins in MVB formation have been shown to be in viral budding E. Sundquist W.I. Annu. Rev. Cell Dev. Biol. 2004; PubMed Google Scholar, A. J. 2004; PubMed Scopus Google Scholar, 2004; 106: PubMed Scopus Google Scholar, A. J. Cell Biol. 2003; PubMed Scopus Google Scholar). in LIP5 in a in HIV-1 viral release. Overexpression of LIP5 with a large epitope not alter the endocytic apparatus as by the of EGF, and or the of a whereas overexpression of proteins with large epitope that are in MVB formation does alter the endocytic apparatus M. B. Ward D.M. E. T. D.M. A. Kaplan J. Sundquist W.I. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, C.R. J. Cell Sci. 2001; PubMed Google Scholar, J. A. D. A. Sci. S. A. 2003; PubMed Scopus Google Scholar, C. H. S. K. M. K. T. Y. M. Biochem. J. 2004; Scholar). Our studies show that LIP5 interacts with The S. cerevisiae homologue of CHMP5 is a class E protein in MVB sorting (21Kranz A. A. R. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). we that the yeast of these two Vta1p and in MVB sorting and also interacted S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). of LIP5 or CHMP5 protein that both proteins play a role in the degradation of the The of silencing these proteins on EGFR is similar to that or proteins that early in MVB sorting, is A. A. Stenmark H. J. Cell Biol. 2003; PubMed Scopus Google Scholar, T. A. C. Stenmark H. Mol. Biol. Cell. 2004; 15: PubMed Scopus Google Scholar). both LIP5 and CHMP5 not result in an on EGFR CHMP5 is a of a large of small proteins (21Kranz A. A. R. Mol. Biol. Cell. 2001; 12: PubMed Scopus Google Scholar). proteins have been identified in and have been identified in yeast (4Katzmann D.J. Odorizzi G. Emr S.D. Nat. Rev. Mol. Cell. Biol. 2002; 3: 893-905Crossref PubMed Scopus (1021) Google Scholar, 10Pornillos O. Garrus J.E. Sundquist W.I. Trends Cell Biol. 2002; 12: 569-579Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). These proteins have a but show only sequence similarity. of to large results in endocytic These are that both early and have been in for cells are with the M.R. A. Luzio J.P. Traffic. 2001; 2: PubMed Scopus Google Scholar). are not MVB proteins are We have two for these is that the large epitope on an protein results in the of different and their These to of vesicles. A is that proteins large epitope into sorting but then in that be and the of sorting of which is these results in data using overexpression or large epitope In S. cerevisiae, Vta1p is to late in MVB but an which the from the MVB (17Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar, 18Shiflett S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In fact, Vta1p has been shown to with Vps4p (17Yeo S.C. Xu L. Ren J. Boulton V.J. Wagle M.D. Liu C. Ren G. Wong P. Zahn R. Sasajala P. Yang H. Piper R.C. Munn A.L. J. Cell Sci. 2003; 116: 3957-3970Crossref PubMed Scopus (82) Google Scholar) and a suggests that interacts with mammalian (19Fujita H. Umezuki Y. Imamura K. Ishikawa D. Uchimura S. Nara A. Yoshimori T. Hayashizaki Y. Kawai J. Ishidoh K. Tanaka Y. Himeno M. J. Cell Sci. 2004; 117: 2997-3009Crossref PubMed Scopus (41) Google Scholar). These results that LIP5 may function in MVB sorting. Depletion of LIP5 and CHMP5 results in similar on EGFR and these are not We that the of depletion of LIP5 and CHMP5 on the of EGFR degradation the distribution of EGFR in LIP5 and CHMP5 cells was cells showed vesicles CHMP5 result may that LIP5 and CHMP5 on different late in the MVB degradation pathway. is that LIP5 and CHMP5 The yeast data that Vta1p interacts with a of the of results in the of Vta1p on the class E compartment, that Vps60p is required for the of Vta1p S.L. Ward D.M. Huynh D. Vaughn M.B. Simmons J.C. Kaplan J. J. Biol. Chem. 2004; 279: 10982-10990Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). on these the mammalian of Vta1p, may The of CHMP5 may affect the of the MVB, in which to a in viral budding The of LIP5 on MVB formation is We to the of the Kaplan for this