Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors

The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p-related sequences. We demonstrate that the Yip1p-related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C-terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family.     

Saccharomyces cerevisiae Rab-GDI displacement factor ortholog Yip3p forms distinct complexes with the Ypt1 Rab GTPase and the reticulon Rtn1p

Rab GTPases are crucial regulators of organelle biogenesis, maintenance, and transport. Multiple Rabs are expressed in all cells, and each is localized to a distinct set of organelles, but little is known regarding the mechanisms by which Rabs are targeted to their resident organelles. Integral membrane proteins have been postulated to serve as receptors that recruit Rabs from the cytosol in a complex with the Rab chaperone, GDI, to facilitate the dissociation of Rab and GDI, hence facilitating loading of Rabs on membranes. We show here that the yeast (Saccharomyces cerevisiae) Golgi Rab GTPase Ypt1p can be copurified with the integral membrane protein Yip3p from detergent cell extracts. In addition, a member of the highly conserved reticulon protein family, Rtn1p, is also associated with Yip3p in vivo. However, Ypt1p did not copurify with Rtn1p, indicating that Yip3p is a component of at least two different protein complexes. Yip3p and Rtn1p are only partially colocalized in cells, with Yip3p localized predominantly to the Golgi and secondarily to the endoplasmic reticulum, whereas Rtn1p is localized predominantly to the endoplasmic reticulum and secondarily to the Golgi. Surprisingly, the intracellular localization of Rabs was not perturbed in yip3Delta or rtn1Delta mutants, suggesting that these proteins do not play a role in targeting Rabs to intracellular membranes. These data indicate that Yip3p may have multiple functions and that its interaction with Rabs is not critical for their recruitment to organelle membranes.     

Specific binding to a novel and essential Golgi membrane protein (Yip1p) functionally links the transport GTPases Ypt1p and Ypt31p.

The regulation of vesicular transport in eukaryotic cells involves Ras-like GTPases of the Ypt/Rab family. Studies in yeast and mammalian cells indicate that individual family members act in vesicle docking/fusion to specific target membranes. Using the two-hybrid system, we have now identified a 248 amino acid, integral membrane protein, termed Yip1, that specifically binds to the transport GTPases Ypt1p and Ypt31p. Evidence for physical interaction of these GTPases with Yip1p was also demonstrated by affinity chromatography and/or co-immunoprecipitation. Like the two GTPases, Yip1p is essential for yeast cell viability and, according to subcellular fractionation and indirect immunofluorescence, is located to Golgi membranes at steady state. Mutant cells depleted of Yip1p and conditionally lethal yip1 mutants at the non-permissive temperature massively accumulate endoplasmic reticulum membranes and display aberrations in protein secretion and glycosylation of secreted invertase. The results suggests for a role for Yip1p in recruiting the two GTPases to Golgi target membranes in preparation for fusion.     

Genetic analysis of yeast Yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor

Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes. The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding. Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes. In this study we have investigated the physiological requirements for Yip1p action. Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability. We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus.     

The Yip1p.Yif1p complex is required for the fusion competence of endoplasmic reticulum-derived vesicles

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.     

At least two regions of the oncoprotein Tre2 are involved in its lack of GAP activity

The product of the human Tre2 oncogene is structurally related to the Ypt/Rab GTPase-activating proteins (Ypt/Rab GAPs). Particularly, the oncoprotein shares with the yeast proteins Msb3p and Msb4p, and with the human protein RN-tre the highly conserved TBC domain, forming the catalytically active domain of Ypt/Rab GAPs. Yet, the Tre2 oncogene seems to encode a nonfunctional Rab GAP. As regions flanking the TBC domain may be crucial for catalytic activity, regions located N- and C-terminally with respect to this domain were explored. For this, chimeric proteins created by sequence exchanges between the Tre2 oncoprotein and RN-tre were tested for their ability to replace functionally the Msb3p and Msb4p proteins in double-mutant yeast cells. These complementation experiments revealed, in addition to the TBC domain, a second Tre2 region involved in the oncoprotein's lack of GAP activity: a 93-aa region flanking the TBC domain on the C-terminal side.     

Identification of Discrete Sites in Yip1A Necessary for Regulation of Endoplasmic Reticulum Structure

The endoplasmic reticulum (ER) of specialized cells can undergo dramatic changes in structural organization, including formation of concentric whorls. We previously reported that depletion of Yip1A, an integral membrane protein conserved between yeast and mammals, caused ER whorl formation reminiscent of that seen in specialized cells. Yip1A and its yeast homologue Yip1p cycle between the ER and early Golgi, have been implicated in a number of distinct trafficking steps, and interact with a conserved set of binding partners including Yif1p/Yif1A and the Ypt1/Ypt31 Rab GTPases. Here, we carried out a mutational analysis of Yip1A to obtain insight into how it regulates ER whorl formation. Most of the Yip1A cytoplasmic domain was dispensable, whereas the transmembrane (TM) domain, especially residues within predicted TM helices 3 and 4, were sensitive to mutagenesis. Comprehensive analysis revealed two discrete functionally required determinants. One was E95 and flanking residues L92 and L96 within the cytoplasmic domain; the other was K146 and nearby residue V152 within the TM domain. Notably, the identified determinants correspond closely to two sites previously found to be essential for yeast viability (E76 and K130 in Yip1p corresponding to E95 and K146 in Yip1A, respectively). In contrast, a third site (E89) also essential for yeast viability (E70 in Yip1p) was dispensable for regulation of whorl formation. Earlier work showed that E76 (E95) was dispensable for binding Yif1p or Ypt1p/Ypt31p, whereas E70 (E89) was required. Collectively, these findings suggest that the ability of Yip1A to bind its established binding partners may be uncoupled from its ability to control ER whorl formation. In support, Yif1A knockdown did not cause ER whorl formation. Thus Yip1A may use the sites identified herein to interact with a novel binding partner to regulate ER membrane organization.     

A role for Yip1p in COPII vesicle biogenesis

Yeast Ypt1p-interacting protein (Yip1p) belongs to a conserved family of transmembrane proteins that interact with Rab GTPases. We encountered Yip1p as a constituent of ER-derived transport vesicles, leading us to hypothesize a direct role for this protein in transport through the early secretory pathway. Using a cell-free assay that recapitulates protein transport from the ER to the Golgi complex, we find that affinity-purified antibodies directed against the hydrophilic amino terminus of Yip1p potently inhibit transport. Surprisingly, inhibition is specific to the COPII-dependent budding stage. In support of this in vitro observation, strains bearing the temperature-sensitive yip1-4 allele accumulate ER membranes at a nonpermissive temperature, with no apparent accumulation of vesicle intermediates. Genetic interaction analyses of the yip1-4 mutation corroborate a function in ER budding. Finally, ordering experiments show that preincubation of ER membranes with COPII proteins decreases sensitivity to anti-Yip1p antibodies, indicating an early requirement for Yip1p in vesicle formation. We propose that Yip1p has a previously unappreciated role in COPII vesicle biogenesis.     

OsPRA1 plays a significant role in targeting of OsRab7 into the tonoplast via the prevacuolar compartment during vacuolar trafficking in plant cells

In yeast and mammals, the Yip/PRA1 family of proteins has been reported to facilitate the delivery of Rab GTPases to the membrane by dissociating the Rab–GDI complex during vesicle trafficking. Recently, we identified OsPRA1, a plant Yip/PRA1 homolog, as an OsRab7-interacting protein that localizes to the prevacuolar compartment, which suggests that it plays a role in vacuolar trafficking of plant cells. Here, we show that OsPRA1 is essential for vacuolar trafficking and that it has molecular properties that are typical of the Yip/PRA1 family of proteins. A trafficking assay using Arabidopsis protoplasts showed that the point mutant OsPRA1_(Y94A) strongly inhibits the vacuolar trafficking of cargo proteins, but has no inhibitory effect on the plasma membrane trafficking of H⁺-ATPase-GFP, suggesting its specific involvement in vacuolar trafficking. Moreover, OsPRA1 was shown to be an integral membrane protein, suggesting that its two hydrophobic domains may mediate membrane integration, and its cytoplasmic N- and C-terminal regions were found to be important for binding to OsRab7. OsPRA1 also interacted with OsVamp3, implying its involvement in vesicle fusion. Finally, we used a yeast expression system to show that OsPRA1 opposes OsGDI2 activity and facilitates the delivery of OsRab7 to the target membrane. Taken together, our results support strongly that OsPRA1 targets OsRab7 to the tonoplast during vacuolar trafficking.     

Yop1p, the yeast homolog of the polyposis locus protein 1, interacts with Yip1p and negatively regulates cell growth

Rab proteins are small GTPases that are essential elements of the protein transport machinery of eukaryotic cells. Each round of membrane transport requires a cycle of Rab protein nucleotide binding and hydrolysis. We have recently characterized a protein, Yip1p, which appears to play a role in Rab-mediated membrane transport in Saccharomyces cerevisiae. In this study, we report the identification of a Yip1p-associated protein, Yop1p. Yop1p is a membrane protein with a hydrophilic region at its N terminus through which it interacts specifically with the cytosolic domain of Yip1p. Yop1p could also be coprecipitated with Rab proteins from total cellular lysates. The TB2 gene is the human homolog of Yop1p (Kinzler, K. W., Nilbert, M. C., Su, L.-K., Vogelstein, B., Bryan, T. M., Levey, D. B., Smith, K. J., Preisinger, A. C., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M. S., Altschul, S. F., Horii, A., Ando, H. M., Y., Miki, Y., Nishisho, I., and Nakamura, Y. (1991) Science 253, 661-665). Our data demonstrate that Yop1p negatively regulates cell growth. Disruption of YOP1 has no apparent effect on cell viability, while overexpression results in cell death, accumulation of internal cell membranes, and a block in membrane traffic. These results suggest that Yop1p acts in conjunction with Yip1p to mediate a common step in membrane traffic.     

Mutations of the SM protein Sly1 resulting in bypass of GTPase requirement in vesicular transport are confined to a short helical region

Ypt/Rab GTPases and Sec1/Munc18 (SM) proteins are key components of the membrane fusion machinery. Here, we describe new mutants of the yeast SM protein Sly1 that specifically bypass the need for GTPases Ypt1 and Ypt6 in vesicular transport. All sequence alterations are confined to a short alpha-helix (alpha-20), which is conserved in fungal Sly1 proteins and, when deleted, results in GTPase suppression. Whereas Sly1p of the evolutionarily distant fission yeast Schizosaccharomyces pombe can functionally replace Sly1p in Sacchromyces cerevisiae, mammalian homologues cannot. This indicates that alpha-20 in fungal Sly1p plays an important role in mediating Ypt/Rab-regulated Sly1p function in membrane fusion.     

An effector of Ypt6p binds the SNARE Tlg1p and mediates selective fusion of vesicles with late Golgi membranes.

Membrane traffic requires vesicles to fuse with a specific target, and SNARE proteins and Rab/Ypt GTPases contribute to this specificity. In the yeast Saccharomyces cerevisae, the Rab/Ypt GTPase Ypt6p is required for fusion of endosome-derived vesicles with the late Golgi. We have shown previously that activation of Ypt6p depends on its exchange factor, Ric1p-Rgp1p, a peripheral membrane protein complex restricted to the Golgi. We show here that a conserved trimeric protein complex, VFT (Vps52/53/54), binds directly to Ypt6p:GTP. Localization of VFT to the Golgi requires Ypt6p, but is unaffected in gos1 and tlg1 mutants, in which late Golgi integral membrane proteins, including SNAREs, are mislocalized. The VFT complex also binds directly to the N-terminal domain of the SNARE Tlg1p, both in vitro and in vivo, in a Ypt6p-independent manner. We suggest that the VFT complex links vesicles containing Tlg1p to their target, which is defined by the local activation of Ypt6p.     

The GTPase-activating enzyme Gyp1p is required for recycling of internalized membrane material by inactivation of the Rab/Ypt GTPase Ypt1p

Rab/Ypt GTPases are key regulators of membrane trafficking and together with SNARE proteins mediate selective fusion of vesicles with target compartments. A family of GTPase-activating enzymes (GAPs) specific for Rab/Ypt GTPases has been discovered, but little is known about their function and substrate specificity in vivo. Here we show that the GAP activity of Gyp1p, a yeast member of this family, is specifically required for recycling of the SNARE Snc1p and the membrane dye FM4-64, implying that inactivation of a Rab/Ypt GTPase may be necessary for recycling of membrane material. Interestingly, recycling of GFP-Snc1p in gyp1 Delta cells is partially restored by reducing the activity of Ypt1p. Moreover, GFP-Snc1p accumulated intracellularly in wild-type cells expressing a GTP-locked, mutant form of Ypt1p (Ypt1p-Q67L), suggesting that GTP hydrolysis of Ypt1p is essential for recycling. Ypt6p is known to be required for the fusion of recycling vesicles to the late Golgi compartment. Interestingly, the deletions of GYP1 and YPT6 were synthetic lethal, raising the possibility that at least two distinct pathways are involved in recycling of membrane material.     

Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.     

Yos1p is a novel subunit of the Yip1p-Yif1p complex and is required for transport between the endoplasmic reticulum and the Golgi complex

Yeast Yip1p is a member of a conserved family of transmembrane proteins that interact with Rab GTPases. Previous studies also have indicated a role for Yip1p in the biogenesis of endoplasmic reticulum (ER)-derived COPII transport vesicles. In this report, we describe the identification and characterization of the uncharacterized open reading frame YER074W-A as a novel multicopy suppressor of the thermosensitive yip1-4 strain. We have termed this gene Yip One Suppressor 1 (YOS1). Yos1p is essential for growth and for function of the secretory pathway; depletion or inactivation of Yos1p blocks transport between the ER and the Golgi complex. YOS1 encodes an integral membrane protein of 87 amino acids that is conserved in eukaryotes. Yos1p localizes to ER and Golgi membranes and is efficiently packaged into ER-derived COPII transport vesicles. Yos1p associates with Yip1p and Yif1p, indicating Yos1p is a novel subunit of the Yip1p-Yif1p complex.     

Overexpression of PRA2, a Rab/Ypt-family small GTPase from Pea Pisum sativum, aggravates the growth defect of yeast ypt mutants

A large number of Rab/Ypt-family small GTPases have been identified from higher plants. While some of them can complement yeast ypt mutants, the expression of Arabidopsis Ara4 protein aggravated the growth defect of a subset of ypt mutants, probably because of the titration of common regulator(s) of yeast Ypt proteins [Ueda, T. et al. (1996) Plant Cell, 8: 2079-20911. PRA2 from pea Pisum sativum encodes an interesting Rab GTPase whose expression is regulated by light [Yoshida, K. et al. (1993) Proc. Natl. Acad. Sci. USA, 90: 6636-6640]. We examined whether PRA2 complements any of the yeast ypt mutants and found again that PRA2 does not complement but rather confers the growth defect to some of the ypt mutants. No growth defect was observed when PRA2 was expressed in the wild-type yeast cells. Unlike the case of Ara4, neither Arabidopsis nor yeast GDI remedied the growth defect by Pra2, indicating that the mechanism of the exacerbation is different. Mutational analysis of PRA2 suggests that the growth inhibition can be ascribed to unidentified factor(s) which prefers the GTP-bound form of Pra2. This yeast system will be useful for identifying such putative regulatory factor(s) from yeast and plants and analyzing their interactions with Pra2.     

The yeast vacuolar Rab GTPase Ypt7p has an activity beyond membrane recruitment of the homotypic fusion and protein sorting-Class C Vps complex

A previous report described lipid mixing of reconstituted proteoliposomes made using lipid mixtures that mimic the composition of yeast vacuoles. This lipid mixing required SNARE {SNAP [soluble NSF (N-ethylmaleimide-sensitive factor)-attachment protein] receptor} proteins, Sec18p and Sec17p (yeast NSF and α-SNAP) and the HOPS (homotypic fusion and protein sorting)-Class C Vps (vacuole protein sorting) complex, but not the vacuolar Rab GTPase Ypt7p. The present study investigates the activity of Ypt7p in proteoliposome lipid mixing. Ypt7p is required for the lipid mixing of proteoliposomes lacking cardiolipin [1,3-bis-(sn-3'-phosphatidyl)-sn-glycerol]. Omission of other lipids with negatively charged and/or small head groups does not cause Ypt7p dependence for lipid mixing. Yeast vacuoles made from strains disrupted for CRD1 (cardiolipin synthase) fuse to the same extent as vacuoles from strains with functional CRD1. Disruption of CRD1 does not alter dependence on Rab GTPases for vacuole fusion. It has been proposed that the recruitment of the HOPS complex to membranes is the main function of Ypt7p. However, Ypt7p is still required for lipid mixing even when the concentration of HOPS complex in lipid-mixing reactions is adjusted such that cardiolipin-free proteoliposomes with or without Ypt7p bind to equal amounts of HOPS. Ypt7p therefore must stimulate membrane fusion by a mechanism that is in addition to recruitment of HOPS to the membrane. This is the first demonstration of such a stimulatory activity--that is, beyond bulk effector recruitment--for a Rab GTPase.     

Primary structure and biochemical characterization of yeast GTPase-activating proteins with substrate preference for the transport GTPase Ypt7p.

Small GTPases of the Ypt/Rab family are regulators of vesicular protein trafficking in exo-and endocytosis. GTPase-activating proteins (GAP) play an important role as down regulators of GTPases. We here report the molecular cloning of a novel GAP-encoding gene (GYP7, for GAP for Ypt7) by high expression from a Saccharomyces cerevisiae genomic library. The GYP7 gene encodes a hydrophilic protein with a molecular mass of 87 kDa. Comparison of its primary sequence with that of the three other known GAPs for transport GTPases, the yeast Gyp6 and Gyp1 proteins and the Rab3A-GAP from rat brain, shows similarity between the yeast GAPs only. Like GYP6 and GYP1, GYP7 is not essential for yeast cell viability. Gyp7p was able to most effectively accelerate the intrinsic GTPase activity of Ypt7p. It was also active, but to a lesser extent, on Ypt31p, Ypt32p and Ypt1p. Ypt6p, Sec4p and the human H-Ras protein did not serve as substrates. We also report the identification and cloning of a gene from the dimorphic yeast Yarrowia lipolytica that encodes a protein whose primary structure and biochemical activity are significantly related to those of Gyp7p from baker's yeast.     

Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p

A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.     

Identification and partial purification of GTPase-activating proteins from yeast and mammalian cells that preferentially act on Ypt1/Rab1 proteins

Two GTPase-activating proteins of apparent molecular mass of 100 kDa and 30 kDa have been partially purified from porcine liver cytosol using mammalian Ypt1/Rab1 protein as substrate. Both proteins act most efficiently on Ypt1/Rab1p, but are inactive with H-Ras p21. From the budding yeast Saccharomyces cerevisiae, a cytosolic 40 kDa yptGAP was partially purified. It accelerates the intrinsic GTPase activity of wild-type Ypt1p but not of H-Ras p21 or a mutant ypt1p with an amino acid substitution of the effector domain which renders the protein functionally inactive in yeast cells.