The Yip1p·Yif1p Complex Is Required for the Fusion Competence of Endoplasmic Reticulum-derived Vesicles

Abstract

Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p·Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery. Here we report that Yip1p and Yif1p, two members of an integral membrane protein complex that bind to the Rab Ypt1p, are required for membrane fusion with the Golgi in vitro. To block fusion, anti-Yip1p or anti-Yif1p antibodies must be added before vesicles bud from the endoplasmic reticulum (ER). These antibodies do not block the packaging of Yip1p, Yif1p, or the soluble NSF attachment protein receptor (SNAREs) into vesicles. We propose that Yip1p and Yif1p perform a critical role in establishing the fusion competence of ER to Golgi vesicles at the time of budding. Consistent with this proposal, we observe that the Yip1p·Yif1p complex binds to the ER to Golgi SNAREs Bos1p and Sec22p, two components of the membrane fusion machinery. The specificity of membrane traffic through the secretory pathway is ensured by the machinery that mediates vesicle tethering, docking, and fusion. The molecular components required for these events are the tethers, Rabs and SNAREs (soluble NSF attachment protein receptor). 1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; NSF, N-ethylmaleimide-sensitive fusion protein; GDI, GDP displacement inhibitor; ER, endoplasmic reticulum; GST, glutathione S-transferase; MBP, maltose binding protein; WT, wild type; PYC, permeabilized yeast cells; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ConA, concanavalin A. The tethers include multisubunit complexes and large coiled-coil proteins, whereas Rabs are key in regulating all tethering events. Tethering factors physically bind the correct carrier vesicle to its appropriate acceptor compartment. This occurs through specific interactions between components on the target membrane and the vesicle (1Whyte J.R. Munro S. J. Cell Sci. 2002; 115: 2627-2637Crossref PubMed Google Scholar). Exactly how the SNAREs contribute to membrane fusion is the subject of an ongoing debate (2Mayer A. Curr. Opin. Cell Biol. 1999; 11: 447-452Crossref PubMed Scopus (79) Google Scholar). It has been demonstrated that purified SNARE proteins inserted into reconstituted liposomes can fulfill a minimal requirement for membrane fusion (3Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Söllner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2049) Google Scholar). However, an open question is whether additional fusion machinery or regulatory factors may contribute to the accelerated fusion rates observed in vivo (4Bruns D. Jahn R. Pflügers Arch. 2002; 443: 333-338Crossref PubMed Scopus (56) Google Scholar). Another unanswered question is how the transition from vesicle tethering to membrane fusion takes place. It is thought that the activation of the Rab is pivotal to the initiation of the downstream docking event, commonly defined as trans-SNARE pairing, which is followed by membrane fusion (5Lian J.P. Stone S. Jiang Y. Lyons P. Ferro-Novick S. Nature. 1994; 372: 698-701Crossref PubMed Scopus (161) Google Scholar, 6Søgaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhausen T. Rothman J.E. Söllner T. Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (447) Google Scholar). Rab proteins belong to a family of small GTPases that regulate membrane traffic (7Novick P. Brennwald P. Cell. 1993; 75: 597-601Abstract Full Text PDF PubMed Scopus (323) Google Scholar). In the cytosol, the GDP-bound form of the Rab is in a complex with guanine nucleotide dissociation inhibitor (GDI). Rabs are recruited to membranes with the aid of a GDI displacement factor (8Dirac-Svejstrup A.B. Sumizawa T. Pfeffer S.R. EMBO J. 1997; 16: 465-472Crossref PubMed Scopus (171) Google Scholar) and inserted into the membrane via a prenyl group. Once the prenylated Rab is delivered to the membrane, it is activated by a specific guanine nucleotide exchange factor through the exchange of GDP for GTP (9Soldati T. Shapiro A.D. Svejstrup A.B. Pfeffer S.R. Nature. 1994; 369: 76-78Crossref PubMed Scopus (155) Google Scholar, 10Ullrich O. Horiuchi H. Bucci C. Zerial M. Nature. 1994; 368: 157-160Crossref PubMed Scopus (255) Google Scholar). Membrane proteins that bind to prenylated Rab proteins have been identified and may provide a clue to the mechanism of Rab attachment to membranes. These factors, which include Yip1p, Yip2p (also called Yop1p), Yip3p, Yip4p, Yip5p, Yif1p, and their mammalian homologs, comprise a family of proteins defined by certain features (11Calero M. Winand N.J. Collins R.N. FEBS Lett. 2002; 515: 89-98Crossref PubMed Scopus (62) Google Scholar). These include a common domain topology with a cytosolic N terminus, luminal C terminus, and multiple transmembrane-spanning domains. Furthermore, these proteins have been shown to interact with multiple Rabs and with each other (11Calero M. Winand N.J. Collins R.N. FEBS Lett. 2002; 515: 89-98Crossref PubMed Scopus (62) Google Scholar, 12Matern H. Yang X. Andrulis E. Sternglanz R. Trepte H.H. Gallwitz D. EMBO J. 2000; 19: 4485-4492Crossref PubMed Scopus (86) Google Scholar, 13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar). Yip1p and Yif1p are the only essential proteins in this family (14Winzeler E.A. Shoemaker D.D. Astromoff A. Liang H. Anderson K. Andre B. Bangham R. Benito R. Boeke J.D. Bussey H. Chu A.M. Connelly C. Davis K. Dietrich F. Dow S.W. El Bakkoury M. Foury F. Friend S.H. Gentalen E. Giaever G. Hegemann J.H. Jones T. Laub M. Liao H. Liebundguth N. Lockhart D.J. Lucau-Danila A. Lussier M. M'Rabet N. Menard P. Mittmann M. Pai C. Rebischung C. Revuelta J.L. Riles L. Roberts C.J. Ross-MacDonald P. Scherens B. Snyder M. Sookhai-Mahadeo S. Storms R.K. Véronneau S. Voet M. Volckaert G. Ward T.R. Wysocki R. Yen G.S. Yu K. Zimmermann K. Philippsen P. Johnston M. Davis R.W. Science. 1999; 285: 901-906Crossref PubMed Scopus (3257) Google Scholar). Yip1p was first identified using the two-hybrid system as a protein that interacts preferentially with the GDP-bound form of Ypt1p and Ypt31p (13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar), two small GTP-binding proteins that act in ER-Golgi and intra-Golgi traffic, respectively (15Bacon R.A. Salminen A. Ruohola H. Novick P. Ferro-Novick S. J. Cell Biol. 1989; 109: 1015-1022Crossref PubMed Scopus (188) Google Scholar, 16Benli M. Doring F. Robinson D.G. Yang X. Gallwitz D. EMBO J. 1996; 15: 6460-6475Crossref PubMed Scopus (142) Google Scholar, 17Jedd G. Mulholland J. Segev N. J. Cell Biol. 1997; 137: 563-580Crossref PubMed Scopus (181) Google Scholar, 18Jedd G. Richardson C. Litt R. Segev N. J. Cell Biol. 1995; 131: 583-590Crossref PubMed Scopus (130) Google Scholar). Yif1p was later identified by its ability to interact with Yip1p in a two-hybrid screen (12Matern H. Yang X. Andrulis E. Sternglanz R. Trepte H.H. Gallwitz D. EMBO J. 2000; 19: 4485-4492Crossref PubMed Scopus (86) Google Scholar). These proteins form a complex and bind to multiple Rabs in yeast (11Calero M. Winand N.J. Collins R.N. FEBS Lett. 2002; 515: 89-98Crossref PubMed Scopus (62) Google Scholar, 12Matern H. Yang X. Andrulis E. Sternglanz R. Trepte H.H. Gallwitz D. EMBO J. 2000; 19: 4485-4492Crossref PubMed Scopus (86) Google Scholar, 13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar). Temperature-sensitive mutations in Yip1p and Yif1p have been shown to block ER-Golgi transport in vivo and lead to the accumulation of ER membranes and 40- to 50-nm vesicles (12Matern H. Yang X. Andrulis E. Sternglanz R. Trepte H.H. Gallwitz D. EMBO J. 2000; 19: 4485-4492Crossref PubMed Scopus (86) Google Scholar, 13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar). Little is known about how the Yip1p family of proteins are involved in membrane traffic. Here we show that the Yip1p·Yif1p complex binds to ER to Golgi SNAREs and is required for the fusion of ER-derived vesicles with the Golgi apparatus. Because the Yip1p family of proteins is highly conserved, it is likely that these family members play a key role in membrane fusion at all stages of vesicle traffic in eukaryotic organisms. Antibody Preparation and Purification—Polyclonal antibodies were raised in rabbits using purified recombinant proteins. Recombinant Yip1p was expressed in bacteria as an N-terminal His tag fusion to amino and purified on to the To purified recombinant Yif1p, an N-terminal fusion to amino was expressed in bacteria using the and purified using were on the appropriate fusion protein to The antibodies on the were and into of anti-Yif1p of purified or protein was to and with were by antibodies with using the to the of and of a Temperature-sensitive the of the the open of was into and into a in which the of been with the and were to the D. R. R. PubMed Scopus Google Scholar) was used to of were used to the open of or of the were was by a the open The was with the into The was by the on minimal and were for on yeast was the of mutations and In transport as as the of and were as J.P. Ferro-Novick S. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, H. Ferro-Novick S. J. Cell Biol. PubMed Scopus Google Scholar). In all was a was on to the used for were an in as J.P. Ferro-Novick S. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). were in the or of of anti-Yif1p or as with the that the protein were with before in To the fusion competence of vesicles in the or of vesicles were to bud from a at before the were a in a Golgi membranes with or were added to the a on to the to were at for were to were to the from in the of The vesicle tethering was as before J. M. Ferro-Novick S. EMBO J. 2000; 19: PubMed Scopus Google Scholar). and were from yeast that been to for or from that been in for to of Yip1p were to and in were a at were at in an for The were in a of to the and all were in for by and were for of of was to a of with and were for with of purified anti-Yif1p The anti-Yif1p antibodies were to a were and with of followed by in of with were In an to was were in and with and with protein followed by to at using an In Yip1p at its N to was expressed in bacteria and purified on as by the fusion proteins were expressed in bacteria and purified on Ypt1p was expressed in yeast as a fusion protein and purified from the membrane as before C. F. 1995; PubMed Scopus Google Scholar). and were purified as before M. R. Biol. 1998; PubMed Scopus Google Scholar). was with nucleotide as Ferro-Novick S. Biol. Cell. 2002; PubMed Scopus Google Scholar). In binding were by of proteins, or with on in a binding CHAPS, GDP in binding using binding with Ypt1p were in binding were at or at for and the were with of binding The protein was in and by using the and ER-Golgi in ongoing in are on how ER-derived transport vesicles to and with the Golgi we to the role of the Yip1p·Yif1p complex in these events. To to this we raised antibodies to Yif1p and Yip1p and their ability to block transport in an in that a of the as a protein to ER-Golgi membrane traffic H. Ferro-Novick S. J. Cell Biol. PubMed Scopus Google Scholar). antibodies to Yif1p transport added to cytosol, Golgi and an system and The ability to transport was antibodies were to whereas was observed the antibodies were to from antibodies transport were with anti-Yip1p antibodies not We fusion was in that anti-Yif1p or anti-Yip1p vesicle was To these we added of anti-Yif1p to and the on was as the of from Membrane fusion was with an that of anti-Yif1p that to fusion, was by or was observed with of In fusion was were with Because of the in vesicle the were to the of from in the of anti-Yif1p and anti-Yip1p antibodies block the stages of ER to Golgi traffic. and to Membrane to the requirement for Yif1p and Yip1p in the of events that in the and tethering of vesicles to the to this question is to the in the in at which transport is to the of The in transport can be into two vesicle and membrane fusion, which can be To the ability of anti-Yif1p and anti-Yip1p antibodies to block fusion added before or vesicles bud from the ER, vesicles were first by with and an at for in the of of was in which was added at the of the were in all and the that the vesicles was in the of were with Golgi membranes and anti-Yif1p or anti-Yip1p in the of antibodies were with Golgi membranes. a on to the antibodies to all fusion were to for we that antibodies Yif1p or Yip1p only fusion added before the with and block in fusion was observed the antibodies were added to vesicles and Golgi membranes with and In to the ER-Golgi Bos1p fusion added before or vesicle not to a of Yip1p and Yif1p is required vesicle budding. Yif1p and Yip1p for ER-derived vesicles to with Golgi membranes must a of events that vesicle tethering, and This that the correct vesicle with the appropriate acceptor compartment. in the of may a block in fusion to a to target or to Golgi membranes. vesicles may to not with Golgi membranes. To between these and to the of the fusion block we a tethering that was before J. M. Ferro-Novick S. EMBO J. 2000; 19: PubMed Scopus Google Scholar). in the or of anti-Yif1p were that ER-derived vesicles from Golgi membranes. vesicles with Golgi membranes J. M. Ferro-Novick S. EMBO J. 2000; 19: PubMed Scopus Google Scholar). The of was by each with with in the of Golgi in to was observed of Golgi The that with the Golgi in anti-Yif1p or anti-Yip1p not were of anti-Yif1p antibodies were This in the of antibodies Yif1p or Yip1p, vesicles not with the Membrane of Ypt1p in the of Yip1p attachment is required for Ypt1p G. Yu J.A. Ferro-Novick S. Nature. PubMed Scopus Google Scholar), and Ypt1p is required for vesicle tethering X. N. C. EMBO J. 1998; 17: PubMed Scopus Google Scholar, R.W. J. Cell Biol. PubMed Scopus Google Scholar). In the yeast two-hybrid Yip1p family members bind preferentially to the prenylated form of Rab proteins (11Calero M. Winand N.J. Collins R.N. FEBS Lett. 2002; 515: 89-98Crossref PubMed Scopus (62) Google Scholar). It has been that these proteins as a membrane or for Rab membrane in that Yip1p and Yif1p are required for vesicle fusion, not tethering, that Ypt1p is not the Yip1p·Yif1p complex is and that Ypt1p may be to membranes. To this is the we the of Ypt1p Yip1p was we a using from wild and a that been to for were at into and and for the of We observed in the of Ypt1p in with In Ypt1p was soluble in from a that is in Ypt1p G. Yu J.A. Ferro-Novick S. Nature. PubMed Scopus Google Scholar). We that the membrane attachment of Ypt1p was not were of was in which the of was the of the to the and as the was for in to of were for the of Ypt1p and in Yip1p not be in the from with Ypt1p with membranes. To the that Ypt1p was or was recruited to the membrane, we were from as and on a used to the ER and Golgi Ypt1p is J. M. Ferro-Novick S. EMBO J. 2000; 19: PubMed Scopus Google Scholar, X. C. J. Cell Biol. 2000; PubMed Scopus Google Scholar). The SNARE Bos1p the Golgi and and the ER and on this proteins are at the of the in the is Because members of a complex are in the of their we for the of shown in the Yif1p Furthermore, the of Ypt1p was in the of the Yip1p·Yif1p complex The was from the were on this not In of the that Ypt1p is required for vesicle tethering, the that Ypt1p membrane is by the of the Yip1p·Yif1p complex in vivo is with the that vesicle tethering occurs in in the of Yip1p or Yif1p The in to a in the of ER-derived specific and packaging of and transport machinery into vesicles from the ER is critical for The of vesicles in the of anti-Yif1p or Yip1p to with Golgi membranes may be the of a to a protein or proteins required for downstream fusion or a of Yif1p or Yip1p to be To to these we the ability of anti-Yif1p or anti-Yip1p to vesicles added before or budding. was used as a in these in the of were in and or in the of and were in the of and and The that were for in vesicle was added before or that the binds to Yif1p or Yip1p on vesicles. Furthermore, these that Yif1p and Yip1p are not physically from vesicles. SNARE proteins are known to be recruited into vesicles and are required for membrane fusion T. S.W. M. H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; PubMed Scopus Google Scholar, S. R. Science. 1998; PubMed Scopus Google Scholar). anti-Yif1p and anti-Yip1p antibodies may block fusion of a to SNAREs into vesicles. To this vesicles were in the or of anti-Yif1p and were vesicles were to a and vesicle were in and for the of shown in the ER to Golgi SNAREs Bos1p and as as Yif1p, were into vesicles in the of anti-Yif1p The membranes and Yif1p were vesicles and not ER, the of these proteins from the was and required Furthermore, a of the ER, was not from the the and were into vesicles not the of vesicles to with Golgi membranes is not to a to ER-Golgi SNAREs or to the of Yif1p or Yip1p from vesicles. ER to Golgi SNAREs to Yip1p in and in that Yip1p and Yif1p are required for membrane fusion. To to the Yip1p·Yif1p machinery interacts with the we the binding of Yip1p for Bos1p in vitro. Bos1p is a that is into vesicles and required for ER-Golgi transport J.P. Ferro-Novick S. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar, J. Ferro-Novick S. Cell. Biol. PubMed Scopus Google Scholar, A. R. J. Cell Biol. 1998; PubMed Scopus Google Scholar). Yip1p was to protein and Because Yip1p has been shown to bind to Ypt1p (13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar), we the ability of to bind to Ypt1p in vitro. and were Ypt1p was purified as a fusion protein from and prenylated Ypt1p was purified from a yeast membrane Because the binding of GDP-bound Ypt1p to is Ypt1p not was with GDP in all binding of of or prenylated specific binding to on Ypt1p to with and was at binding of was not binding to be specific for members of the Rab Because Bos1p is only soluble in the of of which from all binding were with a of the domain of Bos1p that was before S. M. Y. C. Lyons P. A.M. Ferro-Novick S. Biol. Cell. 1997; PubMed Scopus Google Scholar). of to not of the that binding was all SNAREs to binding of was binding of and two SNAREs that act in membrane traffic, was not The in vivo of the binding of SNAREs was by Cell were by and with anti-Yif1p The were for Sec22p, and was in the Bos1p which in Bos1p was was The of Ypt1p and as as Bos1p not was on the of anti-Yif1p of to in a to these proteins the Yip1p·Yif1p complex binds to Bos1p and in for the Yip1p in Membrane Here we have used an in transport that transport between the ER and Golgi to a role for Yip1p and Yif1p in membrane traffic. that vesicles are to in the of Yip1p·Yif1p that these proteins are required for the transition vesicle fusion. between the Yip1p·Yif1p complex and components of the membrane fusion machinery is by the that Bos1p and Sec22p, two ER to Golgi with Yif1p in The requirement for Yip1p proteins in membrane fusion may be common to multiple stages of membrane traffic in all eukaryotic of two Yip1p family Yip1p and Yip3p, have been The of Yip1p, called is to ER and which to the for a role in membrane traffic has from the that the of the of transport of to the in with B. S. R. H. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that Yip1p family members act at other stages of membrane traffic from on the mammalian of Yip3p, called was in two-hybrid as a protein that interacts with multiple Rabs C. M. D. M. 1999; PubMed Scopus Google Scholar, F. J. B. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, G. 2000; PubMed Scopus Google Scholar, J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) in their or GDP-bound It has been that is a GDI displacement factor that to membranes J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In to binding to binds to the SNARE J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). to Golgi membranes M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), it is at the it is a of the it interacts with R. C. B. A.M. S. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), a protein in the of S.H. J. J. EMBO J. PubMed Scopus Google Scholar). question is how the Yip1p family which have been shown to bind to each to membrane fusion at multiple stages of membrane traffic. is that their are and that this may be the from on and its to the has been to be on the The that are for this have been the M. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Yip1p and Yif1p on Golgi membranes at (12Matern H. Yang X. Andrulis E. Sternglanz R. Trepte H.H. Gallwitz D. EMBO J. 2000; 19: 4485-4492Crossref PubMed Scopus (86) Google Scholar, 13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar), it to be whether other family members or interact with SNARE proteins to fusion the The binding of a Rab to the correct membrane is an mechanism in the of each of vesicle The of the Yip1p family of integral membrane proteins as Rab binding proteins to the that may in the of the Rab on membranes (12Matern H. Yang X. Andrulis E. Sternglanz R. Trepte H.H. Gallwitz D. EMBO J. 2000; 19: 4485-4492Crossref PubMed Scopus (86) Google Scholar, 13Yang X. Matern H.T. Gallwitz D. EMBO J. 1998; 17: 4954-4963Crossref PubMed Scopus (112) Google Scholar, J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). we have observed that the Yip1p·Yif1p complex is not required for the membrane of The of Yip1p and Yif1p in vivo not lead to an in the soluble of Ypt1p and Ypt1p is recruited to the correct Consistent with this we that the tethering of ER-derived vesicles to the Golgi is in by the of Yip1p·Yif1p that antibodies to Yip1p or Yif1p must be added vesicle to block fusion that the Yip1p·Yif1p complex is required in the transport at the time a vesicle This may be by that the antibodies may a of Yip1p and Yif1p that is critical for establishing the fusion competence of ER-derived vesicles. Once this has been it be or by the of at a downstream of the the Yip1p·Yif1p complex is required to a that the tethering and fusion machinery to the vesicle at the or it may an factor to the vesicle that is required for fusion. binding of Yip1p·Yif1p to the SNARE complex or trans-SNARE is not we that the of SNARE in from is with wild not However, this must be with to the observed in in in and its to may in for Membrane in and the molecular events that vesicle tethering, the transition into the stages of membrane fusion the of the SNARE T. S.W. M. H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; PubMed Scopus Google Scholar), the that the SNAREs play an role in membrane fusion has a in the membrane to this in with reconstituted liposomes has shown that certain of SNAREs are of membrane fusion J.A. Parlati F. R. Johnston K. F. Söllner T.H. Rothman J.E. Nature. 2000; PubMed Scopus Google Scholar, F. McNew J.A. R. R. Söllner T.H. Rothman J.E. Nature. 2000; PubMed Scopus Google Scholar, F. O. K. McNew J.A. D. Söllner T.H. Rothman J.E. Sci. S. A. 2002; PubMed Scopus Google Scholar). However, an is that SNAREs may play an role in vivo and that additional factors are that downstream of trans-SNARE to the fusion event, by SNARE to the of a fusion C. S. M. A. Nature. PubMed Scopus Google Scholar). that SNAREs may of other fusion machinery C. S. M. A. Nature. PubMed Scopus Google Scholar, C. A. Nature. 1998; PubMed Scopus Google Scholar, C. K. Nature. 1998; PubMed Scopus Google Scholar) and that membrane fusion can of SNARE S. F. A. M. Y. Science. PubMed Scopus Google Scholar). Exactly how SNAREs into this as of membrane fusion or by to downstream to be Because of the of the events that in fusion, it be of to the of the of Yip1p family members with the It is that the Yip1p family of proteins may membrane fusion by regulating their their ability to form trans-SNARE It is that the Yip1p family may a fusion machinery that in with To this it be to Yif1p and Yip1p, or other family are required for fusion at other stages of membrane traffic. We for purified and Brennwald and Novick for and for the We and for