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Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p

2000; Springer Nature; Volume: 19; Issue: 18 Linguagem: Inglês

10.1093/emboj/19.18.4885

1460-2075

Symeon Siniossoglou,

Autophagy in Disease and Therapy

Article15 September 2000free access Ric1p and Rgp1p form a complex that catalyses nucleotide exchange on Ypt6p Symeon Siniossoglou Symeon Siniossoglou Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Sew Y. Peak-Chew Sew Y. Peak-Chew Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Hugh R.B. Pelham Corresponding Author Hugh R.B. Pelham Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Symeon Siniossoglou Symeon Siniossoglou Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Sew Y. Peak-Chew Sew Y. Peak-Chew Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Hugh R.B. Pelham Corresponding Author Hugh R.B. Pelham Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Author Information Symeon Siniossoglou1, Sew Y. Peak-Chew1 and Hugh R.B. Pelham 1 1Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4885-4894https://doi.org/10.1093/emboj/19.18.4885 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cells lacking the GTPase Ypt6p have defects in intracellular traffic and are temperature sensitive. Their growth is severely impaired by additional mutation of IMH1, which encodes a non-essential Golgi-associated coiled-coil protein. A screen for mutants that, like ypt6, specifically impair the growth of imh1 cells led to the identification of RIC1. Ric1p forms a tight complex with a previously uncharacterized protein, Rgp1p. The Ric1p–Rgp1p complex binds Ypt6p in a nucleotide-dependent manner, and purified Ric1p–Rgp1 stimulates guanine nucleotide exchange on Ypt6p in vitro. Deletion of RIC1 or RGP1, like that of YPT6, blocks the recycling of the exocytic SNARE Snc1p from early endosomes to the Golgi and causes temperature-sensitive growth, but this defect can be relieved by overexpression of YPT6. Ric1p largely colocalizes with the late Golgi marker Sec7p. Ypt6p shows a similar distribution, but this is altered when RIC1 or RGP1 is mutated. We infer that the Ric1p–Rgp1p complex serves to activate Ypt6p on Golgi membranes by nucleotide exchange, and that this is required for efficient fusion of endosome-derived vesicles with the Golgi. Introduction A central feature of intracellular membrane traffic is the specific fusion of transport vesicles with their target organelles. Key components of this process are the SNAREs, membrane proteins found on both vesicles and target whose interactions are intimately involved in initiating bilayer fusion. Families of SNARE proteins exist, each with a characteristic distribution, and it is thought that the preferential formation of complexes comprising specific sets of SNAREs contributes to the specificity of membrane fusion (Rothman, 1994; Nichols and Pelham, 1998; Pelham, 1999). Further specificity is provided by additional proteins that aid the initial interaction of vesicles with their targets. These include both tethering factors with coiled-coil motifs and large protein complexes (Pfeffer, 1999; Waters and Pfeffer, 1999). Another set of proteins that may control the initial tethering or docking event are the small GTPases termed rabs (in animal cells) or Ypts (in yeast). Representatives of this family are found at each step of transport (Lazar et al., 1997; Chavrier and Goud, 1999; Gotte et al., 2000). Examples in yeast are Sec4p, required for transport and fusion of exocytic vesicles with the plasma membrane, and Ypt1p, which plays a crucial role in the docking of endoplasmic reticulum (ER)-derived vesicles with early Golgi compartments containing the SNARE Sed5p. Another GTPase associated with the Golgi is Ypt6p, the yeast homologue of mammalian rab6. Unlike Ypt1p, Ypt6p is not required (in most strains) for secretion or for viability, but null mutants have a number of phenotypes including temperature-sensitive growth, partial mis- sorting of the vacuolar protease carboxypeptidase Y and mis-localization of the late Golgi protein Kex2p to the vacuole (Li and Warner, 1996; Tsukada et al., 1999). These phenotypes are quite similar to those of mutants lacking the SNARE Tlg1p, which has been shown to be involved in the recycling of proteins from early endosomes to the Golgi apparatus (Holthuis et al., 1998a,b; Lewis et al., 2000). Hence, a possible role for Ypt6p would be to assist the fusion of endosome-derived vesicles with the late Golgi. A role for Ypt6p in the Golgi is also consistent with some genetic evidence, which suggests a link between the functions of Ypt6p and Ypt1p (Li and Warner, 1998). Despite these clues, rather little is known about the molecules with which Ypt6p might interact. Genetic studies have identified several genes that interact with ypt6, by suppressing its temperature-sensitive growth defect when they are overexpressed (Li and Warner, 1996; Tsukada and Gallwitz, 1996). One such gene was identified as a low-copy suppressor of a ypt6 allele and named IMH1 (Li and Warner, 1996). The same gene was isolated as a high-copy suppressor of a deletion allele of ypt6, and named SYS3 (Tsukada and Gallwitz, 1996); for simplicity we use the name IMH1 here. Since Imh1p has effects even in the absence of Ypt6p, it must be able to act independently of it. However, its function is clearly related, since loss of Imh1p, which normally has little effect on cells, severely reduces the growth of a ypt6 mutant (Tsukada et al., 1999). In this paper we describe a search for proteins interacting with Ypt6p using two independent approaches. In the first, mutations were sought that, like ypt6, show synthetic interactions with imh1. In the second, proteins binding to Ypt6p were identified by protein purification and mass spectrometry. Both approaches led to the identification of a complex consisting of the products of the RIC1 and RGP1 genes. These are peripheral membrane proteins that colocalize with the Golgi marker Sec7p, and we show that they act in vitro as a nucleotide exchange factor for Ypt6p. Their activity does not seem to influence the ability of Ypt6p to bind to membranes, but in their absence the distribution of Ypt6p is distinct from that of the Golgi. We suggest that Ypt6p is able to bind to endosome-derived vesicles, but becomes activated to the GTP-bound form by the Ric1p–Rgp1p complex only at Golgi membranes; Ypt6p–GTP then stimulates the fusion of endosome-derived vesicles with the Golgi. Proteins related to the C-terminal domain of Ric1p are found in a variety of species, including humans. Results Genetic screen for mutants showing synthetic lethality with imh1 Null mutants of YPT6 are viable but temperature sensitive for growth. Though deletion of the IMH1 gene alone has little or no effect on growth, imh1Δ ypt6Δ double mutants show a very severe growth defect (Tsukada et al., 1999). We reasoned that mutations in genes with functions related to that of ypt6 might also show synthetic lethality with imh1, and therefore undertook a screen for such mutants using a colony sectoring assay as described previously (Kranz and Holm, 1990). An imh1Δ yeast strain that carried IMH1 on a plasmid was mutagenized and some 13 000 colonies screened (see Materials and methods). Six mutants were identified that were unable to grow without the plasmid-borne IMH1 gene. Of these, one could be complemented by YPT6, confirming the interaction between imh1 and ypt6. From a yeast genomic library we identified a plasmid that was able to complement four of the remaining mutants; the fifth has proven, so far, refractory to cloning. We restricted the complementary activity of the recovered plasmid to a single intact open reading frame, which corresponded to the RIC1 gene (YLR039c). Figure 1A shows typical data for one of the mutants, sl233. The mutant strain, which lacked the chromosomal copy of IMH1 but carried a URA3 plasmid with the IMH1 gene, was transformed with LEU2 plasmids carrying either RIC1, RIC1–GFP, IMH1 or no inserted gene and the transformants plated on medium containing 5-fluoro-orotic acid (5-FOA) to select cells that had lost the URA3–IMH1 plasmid. As shown in Figure 1, the strain was able to grow only if it contained a plasmid expressing either Imh1p or Ric1p, or the green fluorescent protein (GFP)-tagged version of Ric1p. Figure 1.Functional interaction between IMH1 and RIC1. (A) Synthetic lethal interaction between imh1Δ and ric1 mutants. The sl233 mutant was transformed with the indicated Ycplac111 plasmid (pLEU), either with no insert, or carrying IMH1, RIC1 or a RIC1–GFP fusion. Transformants were grown on plates containing 5-FOA for 3 days at 30°C. (B) Growth properties of the ric1Δ deletion mutant (ric1). Cells from a ric1Δ strain transformed either with an empty LEU2 plasmid or with the same plasmid carrying the wild-type RIC1 gene were diluted in YEPD, spotted on to selective (–Leu) plates and grown at 30 and 37°C for 2 days. Download figure Download PowerPoint RIC1 is a non-essential gene originally isolated in a screen for mutants with altered regulation of ribosome synthesis (Mizuta et al., 1997). This phenotype is common for secretory mutants (Mizuta and Warner, 1994), and indeed the same screen also yielded ypt6 (Li and Warner, 1996). Recently, RIC1 was also identified in a screen for mutants that are synthetically lethal with a temperature-sensitive clathrin heavy chain (Bensen et al., 2000). A role for Ric1p in membrane traffic thus seemed likely, so we characterized it further. Deletion of the RIC1 gene resulted in temperature-sensitive growth, which could be overcome by provision of RIC1 on a plasmid (Figure 1B). We confirmed that a ric1Δ imh1Δ strain was unviable, but found that a ric1Δ ypt6Δ strain was no more defective in terms of growth than a ypt6Δ single mutant (data not shown). The fact that removal of Ric1p had no additional effect once Ypt6p was removed suggests that Ric1p performs a function that is not only related to that of Ypt6p, but is dependent on the presence of Ypt6p. Ric1p binds to Rgp1p To identify proteins that might bind to Ric1p, we expressed a version of the protein tagged at its C-terminus with two IgG-binding domains of protein A (Ric1p–PtA). This was expressed from the RIC1 promoter, on a centromere-containing vector, in ric1Δ cells; it fully complemented the temperature sensitivity of this strain (data not shown). Purification of the fusion protein on IgG–Sepharose revealed a second protein in roughly equimolar amounts (Figure 2, left panel, lane 2). Tryptic digestion of this protein and mass spectrometry of the products (see Materials and methods) showed that it corresponded to Rgp1p, the product of a largely uncharacterized gene reported to be required for efficient growth (Aguilera et al., 1990). Protein A tagging and purification of Rgp1p, performed exactly as for Ric1p, resulted in the copurification of a single protein, which was identified by mass spectrometry as Ric1p (Figure 2, left panel, lane 1). Cleavage of either fusion protein with tobacco etch virus (TEV) protease at a site introduced between the proteins and the protein A tag yielded ∼1:1 molar ratios of Ric1p to Rgp1p, regardless of which was originally tagged (Figure 2, right panel). This indicates that essentially all of the Ric1p and Rgp1p in the cell is in the form of a complex between the two proteins. Figure 2.Ric1p forms a complex with Rgp1p in vivo. (Left) Affinity purification of Rgp1p–PtA and Ric1p–PtA from rgp1Δ and ric1Δ strains, respectively. The purified proteins eluted from the IgG–Sepharose column with low pH were analysed by SDS–PAGE and Coomassie staining. The position of the protein A fusions and the copurifying proteins, identified as Ric1p and Rgp1p by mass spectrometry, are indicated. Molecular mass standards are indicated on the left side of the gel. (Right) The IgG–Sepharose beads carrying the Ric1p–PtA and Rgp1p–PtA fusions were digested by TEV protease and the released fractions containing the cleaved Ric1p and Rgp1p were analysed by SDS–PAGE and Coomassie staining. Molecular mass standards were resolved on the left side of the gel. The position of TEV protease is indicated; the asterisk indicates immunoglobulin heavy chains derived from the affinity column. Download figure Download PowerPoint Deletion of Rgp1p in our standard wild-type strain resulted in a temperature-sensitive growth phenotype very similar to that of ric1Δ cells (data not shown, see also Figure 6). The similar properties of the deletion mutants makes it likely that both proteins are required for the activity of the Ric1p–Rgp1p complex. Ric1p and Rgp1p bind to Ypt6p and act as a nucleotide exchange factor In an independent series of experiments, we expressed a protein A–Ypt6p (PtA–Ypt6p) chimera in yeast cells, under the control of the NOP1 promoter. This fusion protein was functional since it could complement the ypt6 deletion mutant (data not shown). We then isolated PtA–Ypt6p by affinity chromatography on IgG–Sepharose and looked for associated proteins. Since the interactions of the rab/Ypt GTPases are controlled by the state of the bound nucleotide, we incubated a portion of the cell extract with the non-hydrolysable GTP analogue, GTP-γS, prior to purification and performed all subsequent steps in the presence of it. As shown in Figure 3A, two proteins that were present in very low amounts relative to Ypt6p were selectively lost when the extract was incubated with GTP-γS. Mass spectrometry of tryptic digests of these proteins identified them as Ric1p and Rgp1p. To confirm this we co-expressed GFP-tagged Ric1p with PtA–Ypt6p, isolated the Ypt6p and detected Ric1p–GFP by immunoblotting. Figure 3B shows that Ric1p again copurified with Ypt6p, and that it could be released by addition of GTP-γS in the lysis buffer. GDP also released Ric1p, but less efficiently. Thus, we conclude that the Ric1p–Rgp1p complex can bind to Ypt6p, and that it interacts preferentially with the nucleotide-free form and, to a lesser extent, with the GDP form, but not with the GTP form. These binding characteristics are typical for guanine nucleotide exchange factors (GEFs). Figure 3.The Ric1p–Rgp1p complex binds to Ypt6p in vivo in a nucleotide-dependent manner. (A) Affinity purification of a PtA–Ypt6p fusion and identification of proteins that bind to it. A detergent-solubilized supernatant from a ypt6Δ strain expressing a PtA–YPT6 fusion, in the absence (−) or presence of GTP-γS, was loaded onto an IgG–Sepharose column and the purified fusion protein eluted at low pH. Samples from the supernatant loaded on to the column (S) and the eluted PtA–Ypt6p (E), (3700-fold concentrated as compared with the supernatant), were analysed by SDS–PAGE followed by Coomassie staining. The position of PtA–Ypt6p and the copurifying Ric1p and Rgp1p, identified by mass spectrometry, are indicated by asterisks. (B) PtA–Ypt6p was affinity purified from a ypt6Δ ric1Δ double deletion strain co-expressing a Ric1p–GFP fusion from a Ycplac33-URA3 plasmid, in the absence of nucleotide, or in the presence of GDP or GTP-γS. The eluates from the IgG–Sepharose column were analysed by SDS–PAGE followed by western blotting using anti-protein A or anti-GFP antibodies. Download figure Download PowerPoint As a direct test of GEF activity, we purified Ypt6p from a yeast strain lacking both Rgp1p and Ric1p (see Materials and methods), loaded it with [3H]GDP, incubated it with excess unlabelled GDP and monitored the release of labelled GDP from the protein. As shown in Figure 4A, the spontaneous rate of release was low but addition of the Ric1p–Rgp1p complex, purified by protein A affinity chromatography and TEV protease cleavage as in Figure 2, accelerated release in a dose-dependent manner. Identical results were obtained when unlabelled GTP was used as competitor (data not shown). This effect was clearly catalytic, since complete release of [3H]GDP from 90 pmol Ypt6p could be induced by only 8 pmol of the complex; only 20% of the [3H]GDP was released without the complex within the same time period. Figure 4.The Ric1p–Rgp1p complex stimulates GDP exchange on Ypt6p. (A) Ypt6p and the Ric1p–Rgp1p complex were purified from yeast as described in Materials and methods. Ninety picomoles of Ypt6p pre-incubated with [3H]GDP were incubated with the indicated amounts of Ric1p–Rgp1p at 24°C, and protein-bound radioactivity was determined at intervals by filtration of aliquots through nitrocellulose. Background binding (with bovine serum albumin in place of Ypt6p) was equivalent to ∼10% of the total and has not been subtracted. (B) Ypt1p preloaded with [3H]GDP was incubated with the Ric1p–Rgp1p complex at 24°C and nucleotide exchange was determined as in (A). (C) Ypt6p preloaded with [3H]GDP was incubated independently with either Ric1p or Rgp1p or the Ric1p–Rgp1p complex at 24°C. Nucleotide exchange was determined as in (A). Download figure Download PowerPoint The exchange activity was specific, since no effect of Ric1p–Rgp1p was seen when Ypt1p was used in place of Ypt6p (Figure 4B). We also tested Ric1p and Rgp1p separately, expressing each from a multicopy vector in the appropriate deletion strain. This was necessary because Rgp1p, in particular, appeared unstable in the absence of its partner. However, neither protein alone provided detectable exchange activity (Figure 4C). Taken together these data show that the Ric1p–Rgp1p complex can bind to Ypt6p and function as an exchange factor. Like other exchange factors, it probably acts primarily by destabilizing the Ypt6p–GDP complex. Role of Ric1p–Rgp1p in Ypt6p-mediated traffic If Ric1p and Rgp1p are important for the formation of Ypt6p–GTP, the presumed active form of the protein, then one would expect cells lacking one or both to have phenotypes similar to those of ypt6 mutants (Li and Warner, 1996; Tsukada et al., 1999). Indeed, like ypt6 cells, a ric1 mutant showed little defect in secretion of invertase or transport to the plasma membrane of an endocytosis-defective version of the exocytic SNARE Snc1p (data not shown), and only a mild mis-sorting of the vacuolar protease carboxypeptidase Y (Bensen et al., 2000). ypt6, ric1 and rgp1 cells all showed a similar characteristic fragmentation of vacuoles, as visualized by uptake of the dye FM4-64 (Figure 5A). Since many of the phenotypes described for ypt6 are similar to those of tlg1, a gene required for recycling of proteins such as Snc1p through early endosomes to the Golgi, we also examined the distribution of GFP–Snc1p in the various mutants. In wild-type cells this protein travels from Golgi to plasma membrane, is endocytosed and returns to the Golgi for re-use (Lewis et al., 2000); in the steady state it is visible on the plasma membrane and in internal dots corresponding to endosomes and/or Golgi cisternae. In ypt6, ric1 and rgp1 cells it was entirely intracellular, in an indistinct hazy or dotty pattern that probably corresponds at least in part to transport vesicles that are unable to fuse with their target (Figure 5B). Hence, as previously proposed for Ypt6p (Tsukada et al., 1999), all three proteins are likely to be involved in endosome–Golgi recycling. Figure 5.Defects in vacuolar morphology and GFP–Snc1p recycling in ric1Δ, rgp1Δ and ypt6Δ mutants. (A) The ric1Δ, rgp1Δ and ypt6Δ deletion mutants and a wild-type strain were grown at 30°C to exponential phase, labelled with the endocytic tracer dye FM4-64 for 15 min and chased for 1 h. Note that only vacuoles are visible under these conditions. (B) The deletion mutants were transformed with a plasmid expressing GFP–Snc1p. Transformants were grown to exponential phase at 30°C and inspected by confocal microscopy. Download figure Download PowerPoint Despite these similarities, we had observed a more severe growth defect for ypt6Δ cells than for ric1Δ, rgp1Δ or a ric1Δ rgp1Δ double mutant, especially on selective medium at elevated temperatures. This implied that Ypt6p was capable of some function even without the GEF activity provided by Ric1p and Rgp1p. In confirmation of this, we found that even very mild overexpression of Ypt6p (from a low-copy plasmid and the YPT6 promoter) was sufficient to improve growth of ric1Δ, rgp1Δ and the double mutant at 37°C, and when a stronger promoter was used to drive YPT6 expression, growth was restored to wild-type levels (Figure 6). This could be accounted for by a low level of Ypt6p–GTP being generated spontaneously, or by the action of some other GEF. It strongly suggests that the major role of Ric1p and Rgp1p in vivo is to activate Ypt6p. Conversely, we can conclude that once activated by nucleotide exchange, Ypt6p does not require Ric1p or Rgp1p to stimulate membrane traffic. Figure 6.YPT6 suppresses the temperature-sensitive growth defect of the ric1, rgp1 and ric1 rgp1 deletion mutants. The ric1, rgp1 and ric1 rgp1 deletion mutants were transformed with an empty centromeric plasmid (YCplac111), YCp plasmid(s) carrying the complementing gene(s) as indicated, or with a centromeric plasmid over-expressing PtA–YPT6 [pUN100-(PNOP1)PtA-YPT6]. Transformants were spotted onto selective (–Leu) plates and incubated for 2 days at 37°C. Download figure Download PowerPoint Location of Ypt6p and Ric1p–Rgp1p in cells We used functional GFP chimeras, expressed at endogenous levels in the absence of the corresponding untagged protein, to localize Ypt6p, Ric1p and Rgp1p. All of these fusion proteins retained their function (Figure 1A and data not shown). None are integral membrane proteins, but confocal microscopy showed that each was present in a dotted pattern typical of the yeast Golgi (Figure 7A). To identify the dots, we colocalized Ric1p–GFP with a YFP-tagged version of Sec7p (Figure 7; see Materials and methods for details). Analysis of fields of cells showed that 75–80% of the Ric1p dots also contained Sec7p. The dots containing the two proteins sometimes showed slight differences in shape or position, suggesting segregation of the proteins within a single structure. Since Sec7p is a late Golgi marker (Rossanese et al., 1999; Lewis et al., 2000), the colocalization confirms that the Ric1p–Rgp1p complex is present on Golgi membranes. A similar analysis showed that Ypt6p also overlapped with Sec7p (Figure 7), but interestingly, the degree of overlap appeared lower: only 50–60% of the Ypt6p dots contained Sec7p. Hence, Ypt6p may not be restricted to late Golgi membranes alone. Figure 7.Localization of Ric1p–GFP, Rgp1p–GFP and GFP–Ypt6p fusions. (A) RIC1–GFP, RGP1–GFP and GFP–YPT6 were expressed from centromeric plasmids under the control of their own promoters, in the corresponding deletion mutants. (B) The Ric1p–GFP and GFP–Ypt6p strains were mated with a strain bearing a chromosomal copy of YFP–SEC7, under the control of the TPI1 promoter. YFP and GFP images of diploid cells were obtained as described in Materials and methods. Arrows indicate identical positions in the two images. Download figure Download PowerPoint Ric1p–Rgp1p affects the localization of Ypt6p We next examined Ric1p–GFP and GFP–Ypt6p individually in a ypt6Δ ric1Δ strain; since the GFP-tagged proteins are functional, this allowed the effects of removal of one on the distribution of the other to be tested. As might be expected, since the Golgi complex remains functional, the dotty pattern of Ric1p–GFP was unchanged in ypt6 cells (Figure 8A). This shows that binding of the Ric1p–Rgp1p complex to membranes does not require Ypt6p. However, removal of Ric1p had a dramatic effect on the distribution of GFP–Ypt6p: the pattern changed from dots (when RIC1 was provided on a plasmid) to an even haze (Figure 8B). Similar results were obtained with an rgp1 deletion mutant (data not shown). Since removal of Ric1p is not likely to have any greater effect on Golgi structure than removal of Ypt6p, the absence of a dotty pattern for GFP–Ypt6p suggests that this protein is absent from Golgi membranes in a ric1 mutant. Figure 8.Subcellular distribution of GFP–Ypt6p is drastically altered in ric1 deletion mutants. (A) The ypt6 ric1 double deletion mutant was transformed with a centromeric plasmid expressing Ric1p–GFP and examined under the confocal microscope. (B) A ypt6 ric1 double deletion mutant transformed with the indicated plasmids was examined under the MRC-600 confocal microscope. (C) The GFP–YPT6 plasmid was transformed into ypt6 cells, thus complementing them ('wild-type') or into the double ypt6 ric1 deletion mutant ('ric1') and fractionated into 13 000 and 100 000 g pellets (P13 and P100, respectively) and 100 000 g supernatant (S100). Equivalent amounts of each fraction were analysed by SDS–PAGE followed by western blotting with anti-GFP, anti-Pgk1p and anti-Tlg1p antibodies. The upper band visible in the S100 fraction probed with anti-GFP is a cross-reacting endogenous protein. Download figure Download PowerPoint A possible explanation would be that only the GTP-bound form of Ypt6p associates with membranes. To test this, we fractionated the cells by lysis and centrifugation at 13 000 g and then 100 000 g and analysed the pellets and supernatant by immunoblotting (Figure 8C). As controls, we checked the soluble enzyme phosphoglycerokinase (Pgk1p), which was found primarily in the supernatant, and the late Golgi/early endosomal SNARE Tlg1p, which was found partly in the P13 fraction and partly in the P100 fraction. In wild-type cells, GFP–Ypt6p was distributed between all three fractions, with ∼80% being present in the combined membrane pellets and the remainder in the supernatant. Strikingly, however, this distribution was almost identical whether or not Ric1p was present. Thus, the change in distribution of GFP–Ypt6p is unlikely to be due to its release from membranes. To summarize, in the absence of its exchange factor complex, Ypt6p changes its punctate Golgi localization to a haze, yet appears to be still membrane bound. It is possible that in a ric1 mutant, Ypt6p is bound to small endosome-derived transport vesicles, which, because they are numerous and scattered, appear as a haze. This could occur if Ypt6p binds initially to endosomal membranes and is delivered to the Golgi on vesicles, or if it is permanently membrane associated and cycles through early endosomes in a similar manner to resident late Golgi membrane proteins. By the same argument, the fact that the distribution of Ric1p is unaltered in ypt6Δ cells implies that it does not cycle through early endosomes. Discussion In this paper we have shown that Ric1p and Rgp1p form a complex that is peripherally associated with Golgi membranes and that has the ability to catalyse, in vitro, guanine nucleotide exchange on Ypt6p. This in vitro evidence is supported by the observations that small amounts of Ric1p and Rgp1p can be isolated from cells in a complex with Ypt6p, which dissociates upon addition of GTP. Furthermore, ric1 and rgp1 mutants have similar phenotypes to a ypt6 mutant, and like ypt6Δ, a ric1Δ mutant shows synthetic lethality with an imh1Δ mutation. A ric1 mutant was originally isolated in a screen for genes affecting ribosome synthesis, and Ric1p was postulated to be a transcription factor (Mizuta et al., 1997). However, the same screen yielded a ypt6 mutant, and it has been established that a variety of secretory mutants affect ribosome synthesis (Mizuta and Warner, 1994; Li and Warner, 1996). RIC1 also shows genetic interactions with clathrin mutants (Bensen et al., 2000). Hence, the effects of Ric1p on ribosome synthesis are likely to be secondary to its effects on Ypt6p. Removal of Ric1p from a ypt6Δ strain does not result in any additional growth impairment, and the growth defects of ric1 and rgp1 mutants can be fully rescued by overexpression of YPT6. This argues that the major and perhaps only role of Ric1p and Rgp1p is in the activation of Ypt6p. Why two proteins should be required for GEF function is unclear. However, the single proteins seem relatively unstable, and preliminary results indicate that neither Ric1p nor Rgp1p is able to localize to Golgi membranes efficiently in the absence of its partner. Thus, the complex forms a functional unit. Further analysis will be required to determine the precise residues responsible for the GEF activity. Neither Ric1p nor Rgp1p shows any detectable homology to other known GEFs, including those active on rab-like GTPases such as Sec2p (a GEF for Sec4p), Vps9p (a GEF for Vps21p) or the rab5 GEF rabex 5. However, database searches show that Ric1p contains a C-terminal region that is clearly homologous to proteins in Schizosaccharomyces pombe and other species, including humans (Figure 9). The proteins from higher eukaryotes that contain this domain are obvious candidates for rab6 exchange factors. Figure 9.Multiple sequence alignment of the C-terminal domain of Ric1p with related sequences found in proteins from other species: Drosophila melanogaster (DDBJ/EMBL/GenBank accession No. AF181626.1); Homo sapiens (AB037853.1; brackets in the residue number indicate that it is a partial sequence); Caenorhabditis elegans (Q09