Portal de Periódicos da CAPES

Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes

2003; Springer Nature; Volume: 22; Issue: 3 Linguagem: Inglês

10.1093/emboj/cdg062

1460-2075

Ewald H. Hettema, Michael Lewis, Michael W. Black, Hugh R.B. Pelham,

Microtubule and mitosis dynamics

Article3 February 2003free access Retromer and the sorting nexins Snx4/41/42 mediate distinct retrieval pathways from yeast endosomes Ewald H. Hettema Ewald H. Hettema MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Michael J. Lewis Michael J. Lewis MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Michael W. Black Michael W. Black Present address: California Polytechnic State University, Biological Sciences Department, San Luis Obispo, CA, 93407 USA Search for more papers by this author Hugh R.B. Pelham Corresponding Author Hugh R.B. Pelham MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Ewald H. Hettema Ewald H. Hettema MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Michael J. Lewis Michael J. Lewis MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Michael W. Black Michael W. Black Present address: California Polytechnic State University, Biological Sciences Department, San Luis Obispo, CA, 93407 USA Search for more papers by this author Hugh R.B. Pelham Corresponding Author Hugh R.B. Pelham MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK Search for more papers by this author Author Information Ewald H. Hettema1, Michael J. Lewis1, Michael W. Black2 and Hugh R.B. Pelham 1 1MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH UK 2Present address: California Polytechnic State University, Biological Sciences Department, San Luis Obispo, CA, 93407 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:548-557https://doi.org/10.1093/emboj/cdg062 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The endocytic pathway in yeast leads to the vacuole, but resident proteins of the late Golgi, and some endocytosed proteins such as the exocytic SNARE Snc1p, are retrieved specifically to the Golgi. Retrieval can occur from both a late pre-vacuolar compartment and early or ‘post-Golgi’ endosomes. We show that the endosomal SNARE Pep12p, and a mutant version that reaches the cell surface and is endocytosed, are retrieved from pre-vacuolar endosomes. As with Golgi proteins, this requires the sorting nexin Grd19p and components of the retromer coat, supporting the view that endosomal and Golgi residents both cycle continuously between the exocytic and endocytic pathways. In contrast, retrieval of Snc1p from post-Golgi endosomes requires the sorting nexin Snx4p, to which Snc1p can be cross-linked. Snx4p binds to Snx41p/ydr425w and to Snx42p/ydl113c, both of which are also required for efficient Snc1p sorting. Our findings suggest a general role for yeast sorting nexins in protein retrieval, rather than degradation, and indicate that different sorting nexins operate in different classes of endosomes. Introduction Elucidating the ways in which proteins are localized to particular cellular compartments is a crucial step in understanding cellular organization. The organelles of the endomembrane system present a particular challenge. They are connected by a complex network of vesicular trafficking pathways, and the steady-state distribution of membrane proteins between them is determined by the kinetics with which these proteins move along the various pathways. The routes the proteins follow can be deduced from the effects of mutations, in particular those that disrupt sorting signals required for entry into a particular pathway. A major goal, therefore, is to define the organelles and pathways, the sorting signals on proteins and the machinery that recognizes them. In yeast cells, the endocytic pathway leads to the vacuole, which shares the degradative properties of animal cell lysosomes. Once delivered to endosomes, from either the plasma membrane or the Golgi apparatus, integral membrane proteins require no particular cytoplasmic signals to proceed to the vacuole. Thus, removal of the cytoplasmic tails from proteins that reside in endosomes or the late Golgi results in their transfer to the vacuole. Such proteins are normally saved from this fate by a specific retention or retrieval process, and much evidence suggests that they are recycled actively from endosomes to the Golgi (reviewed by Conibear and Stevens, 1998). The retrieved proteins include receptors and enzymes that cycle between the Golgi and endosomes, and some endocytosed proteins such as the SNARE Snc1p and the chitin synthase Chs3p (Conibear and Stevens, 1998; Holthuis et al., 1998; Lewis et al., 2000; Valdivia et al., 2002). At least two distinct retrieval pathways have been identified. One originates in early or ‘post-Golgi’ endosomes (PGEs; Pelham, 2002), and is the route followed normally by Snc1p, Chs3p and probably by the late Golgi SNAREs Tlg1p and Tlg2p (step 6 in Figure 1) (Holthuis et al., 1998; Lewis et al., 2000; Valdivia et al., 2002). Another comes from late or ‘pre-vacuolar’ endosomes (PVEs; step 7), and is the pathway used by the carboxypeptidase Y sorting receptor Vps10p (Piper et al., 1995). These pathways can be distinguished in class E vacuolar protein sorting (vps) mutants, which accumulate multilamellar structures corresponding to abnormally enlarged PVEs (Rieder et al., 1996; Babst et al., 1997). In such mutants, retrieval from PVEs is inefficient, but recycling from PGEs continues (Lewis et al., 2000). However, many proteins, including Chs3p and the late Golgi proteins Kex2p and Ste13p, appear to carry multiple sorting signals that allow them to travel via either route, which complicates analysis (Brickner and Fuller, 1997; Bryant and Stevens, 1997; Valdivia et al., 2002). Figure 1.Trafficking pathways discussed herein. From the late Golgi, proteins can pass to the plasma membrane (1) or to post-Golgi endosomes (PGEs) (2) depending on the nature of their TMDs, or via the selective GGA protein-dependent pathway to pre-vacuolar endosomes (PVEs) (3). The FSD motif is required for Pep12p to follow this route. Proteins in PGEs can also pass, without special signals, to PVEs (4) and then to the vacuole. Snc1p is endocytosed to PGEs (5), then retrieved to the Golgi (6) by a mechanism that requires Snx4p. Proteins can also be retrieved from PVEs (7), a step that for Pep12p requires Grd19p. This is also the step mediated by retromer. Other pathways exist but have been omitted for clarity. Download figure Download PowerPoint Figure 2.Mutants that affect retrieval of Pep12p. (A) GFP–Pep12p bearing the Sso1p TMD and a mutant FSD signal (F20L) is in punctate PGE structures with either the F6L or I71T mutations, but with both it is on vacuolar membranes. (B) Sucrose gradient fractionation of Δpep12 cells expressing FSD− Pep12p, with its own TMD, with or without the F6L/I71T mutations. Fraction 1 is the top of the gradient. The positions of PGEs and vacuolar membranes, identified by the presence of Tlg1p and Vam3p, respectively (Black and Pelham, 2000), are indicated. PVEs, defined by Pep12p itself, run in the centre of the gradient. Note that the extracts were pre-cleared by medium-speed centrifugation, resulting in substantial under-representation of vacuoles on the gradients. (C) Quantitation of the indicated proteins in gradient fractions. The left hand panel corresponds to the gradients in (B). Download figure Download PowerPoint Signal-dependent sorting is usually the hallmark of protein coats, which gather particular subsets of proteins into transport carriers. There is evidence that two different kinds of clathrin coat can be found on endosomal membranes, at least in animal cells (Raiborg et al., 2002; Sachse et al., 2002), and also that COPI contributes to endosomal sorting (Daro et al., 1997; Piguet et al., 1999), but it remains unclear whether either clathrin or COPI is directly involved in retrieval in yeast. Both types of coat are also present on Golgi membranes, where they perform different sorting functions (Scales et al., 2000; Black and Pelham, 2001). Thus, even if the same coats are used, the cargo selection mechanism is likely to differ between Golgi and endosome. One putative coat complex that seems to be involved specifically in retrieval from endosomes has been identified, namely the retromer. This consists of five proteins; Vps5p and Vps17p form a dimer, which associates with a complex of Vps26p, Vps29p and Vps35p (Seaman et al., 1998). All of these proteins are required for recycling of Vps10p from PVEs, but not for the retrieval of Snc1p from PGEs (Lewis et al., 2000). The retromer helps to retrieve a variety of late Golgi proteins, and there is good evidence that Vps35p recognizes sorting signals on the cytoplasmic tails of Ste13p and Vps10p (Nothwehr et al., 1999, 2000). Vps5p and Vps17p are examples of sorting nexins, peripheral membrane proteins originally identified in animal cells as proteins that bind to the cytoplasmic portions of receptor tyrosine kinases such as the epidermal growth factor receptor (Kurten et al., 1996). The functions of the sorting nexins have been somewhat unclear; they were postulated to mediate the down-regulation and degradation of receptors in animal cells, but the yeast data suggest instead that they may rescue proteins from degradation. Sorting nexins can be defined as proteins that have a PX domain and a role in the sorting of membrane proteins. PX domains recently have been shown to bind lipids and to have specificity, in most cases, for the endosomal lipid phosphatidylinositol 3-phosphate (PI3P) (Bravo et al., 2001; Yu and Lemmon, 2001; for reviews, see Misra et al., 2001; Sato et al., 2001). Of the 15 PX domain proteins in yeast, five have known functions that do not involve cargo sorting in endosomes (Vam7p, Spo14p, Mdm1p, Bem1p and Bem3p). Besides the retromer components, two others have endosomal functions: Mvp1p is required for the efficient retrieval of Kex2p and Vps10p from endosomes (Chang and Fink, 1995; Ekena and Stevens, 1995), while Grd19p affects sorting of Kex2p and Ste13p, but not Vps10p (Voos and Stevens, 1998). The remaining proteins are largely uncharacterized, but all are candidates for additional sorting nexins. In this study, we explore the roles of sorting nexins in the trafficking of two proteins that undergo endocytosis and then pass to the Golgi: the exocytic SNARE Snc1p and a mutated version of the endosomal SNARE Pep12p. We show that sorting of both the mutant Pep12p and of the endogenous wild-type protein is strongly dependent on Grd19p and retromer, which act in PVEs. In contrast, the sorting nexins Snx4p, Ydr425p (Snx41p) and Ydl113p (Snx42p) are required for recycling of Snc1p from PGEs. Thus, distinct sets of sorting nexins mediate retrieval from the two main types of yeast endosome. Snx4p and its companions are the clearest examples so far identified of proteins required for sorting in PGEs. Results Pep12p mutants that fail to be retrieved from endosomes Pep12p normally travels from Golgi membranes to PVEs by a clathrin- and GGA protein-dependent pathway (step 3 in Figure 1). The wild-type protein is found in PVEs, but the localization mechanism is saturable: when overexpressed, a green fluorescent protein (GFP)-tagged version of Pep12p is found almost exclusively on the vacuolar membrane (Black and Pelham, 2000). By the introduction of appropriate mutations, Pep12p can be routed instead to PGEs either directly (step 2 in Figure 1) or via the plasma membrane (steps 1 and 5). In particular, the plasma membrane route is followed by a GFP-tagged derivative of Pep12p bearing the Sso1p transmembrane domain (TMD) and a mutation in the FSD motif that is required for sorting into the GGA pathway (Black and Pelham, 2000). In the steady state, this protein shows a punctate distribution that corresponds largely to PGEs, together with some plasma membrane fluorescence (Black and Pelham, 2000; see below, Figure 3A). We chose to study this version of Pep12p, since it successfully avoids accumulation in the vacuole and could thus yield information on how endocytosed proteins avoid this fate. Figure 3.Distribution of Pep12 derivatives in various mutants. (A) GFP–Pep12p is in punctate PGEs in wild-type cells (WT), whether it is delivered there directly from the Golgi (FSD−) or via the plasma membrane (FSD−, Sso1 TMD), but both constructs are on vacuolar membranes in grd19 cells. (B) Distribution of GFP-tagged FSD− Pep12p in deletion mutants of the nine sorting nexin mutants indicated. (C) Immunoblotting of endogenous Pep12p in medium-speed (p13) and high-speed pellet fractions (p100) of the indicated strains. (D) GFP–Pep12p in a vps4 temperature-sensitive mutant grown overnight at 25°C (only vacuoles are visible) or 37°C (pre-vacuolar structures). For the right hand panels, the protein was expressed from the GAL1 promoter for 5 h, expression stopped by shifting to glucose medium for 2 h, then the temperature shifted from 25 to 37°C and cells imaged 2 and 4 h later. Download figure Download PowerPoint We randomly mutagenized the coding sequence of this Pep12p derivative by PCR, and selected variants that showed a vacuolar pattern by fluorescence microscopy. Two such mutants were analysed further. Both contained multiple mutations, but the phenotypic effect could be ascribed to those near the N-terminus. In one case, the effect was due to two simultaneous changes, F6L and I71T. Neither mutation alone was sufficient for mistargeting (Figure 2A), and indeed deletion of the first 20 residues of the protein also had no effect (data not shown). This suggests that either a patch on the surface of the protein is recognized, rather than a simple linear sequence, or the mutations have an indirect effect on the structure of the protein. The second mutant contained seven changes between positions 31 and 93, but we did not investigate these in detail. We next examined the effects of the F6L/I71T mutations on Pep12p itself, expressed at wild-type levels in a pep12-null mutant. Initially, we continued to use a version of the protein mutated in the FSD motif, but with its own TMD. This protein is delivered directly from the Golgi to PGEs, without passing to the plasma membrane (step 2 in Figure 1; Black and Pelham, 2000). Even with the F6L/I71T changes, it complemented the growth and trafficking defects of the pep12 cells, indicating that it remained functional. Extracts from cells expressing Pep12p with or without the retrieval mutations were fractionated, and endosomal membranes remaining in the supernatant after centrifugation at 13 000 g were separated on sucrose density gradients (Figure 2B). On these gradients, PGE markers are found close to the bottom, while PVEs are spread through the centre; any vacuoles remaining after the 13 000 g centrifugation float to the top (Black and Pelham, 2000). The F6L/I71T mutations caused dramatic loss of Pep12p from PVEs, with a concomitant increase in the vacuolar fractions but, interestingly, loss from the PGE region was much less severe (Figure 2B and C). When the experiment was repeated using a version of Pep12p with the FSD motif intact, thus reducing delivery to PGEs and directing the protein instead to PVEs, the F6L/I71T mutations caused complete transfer to the vacuole (Figure 2C). Thus, although we had screened for loss from PGEs, the mutations we obtained affect sorting at the level of PVEs. Loss from PGEs may be secondary to this, reflecting a slow but steady delivery to PVEs. Grd19p and retromer are required for sorting of Pep12p To search for proteins responsible for the sorting of Pep12p, we systematically examined sorting nexin mutants. This approach revealed that Grd19p was required for retrieval of the GFP-tagged FSD− variant of Pep12p; in a grd19 mutant, the GFP pattern changed from punctate to vacuolar (Figure 3A). Similar results were obtained whether the construct had the Pep12p TMD or the Sso1p TMD, i.e. whether it reached PGEs from the Golgi or from the plasma membrane. Mutations in the retromer components Vps5p and Vps17p also affected the distribution of Pep12p, but the results were less clear, in part because vps5 and vps17 mutants have fragmented vacuoles. Removal of any of the other seven putative sorting nexins had no obvious effect on the distribution of tagged Pep12p (Figure 3B). To confirm these results, we fractionated cells by differential centrifugation and examined the distribution of endogenous Pep12p, which is normally distributed between the 13 000 g pellet (p13) and the 100 000 g pellet (p100). Figure 3C shows that in grd19, vps5 and vps17 cells, Pep12p was almost exclusively in p13, where the vacuoles are found. Similar results were obtained with another retromer mutant, vps35 (data not shown). In contrast, the Pep12p distribution was unaltered in another sorting nexin mutant, snx4 (Figure 3C). Surprisingly, despite their mistargeting of Pep12p, grd19 cells have a normal vacuolar morphology and sort vacuolar hydrolases efficiently. This is, however, consistent with previous observations that quite low levels of Pep12p are sufficient to maintain endosomal function (Reggiori et al., 2000). Interestingly, in grd19 cells and retromer mutants, a minor form of Pep12p with an apparent size 2–3 kDa larger than normal was present (Figure 3C). A similar minor form could also be detected with the F6L/I71T mutant protein, in both the vacuolar and PGE fractions (Figure 2B). The nature of this apparent modification is unclear; preliminary experiments showed little effect of phosphatase treatment, suggesting that it does not represent phosphorylation. The modification correlates with mis-sorting, but whether it contributes to this, or is merely a reflection of the altered state of Pep12p, remains to be determined. Previously, retromer and Grd19p have been implicated in the recycling of proteins from PVEs to the Golgi, and it seems likely that Pep12p follows this same route. However, it has been reported that an artificial construct bearing both a vacuole targeting signal and Golgi retrieval signal can reach the pre-vacuolar compartment of a class E vps mutant via the vacuole (Bryant et al., 1998). Such a vacuole–endosome pathway could, in principle, also help to maintain Pep12p in endosomes. To test this, we overexpressed GFP–Pep12p in a temperature-sensitive class E mutant, vps4 (Babst et al., 1997). As expected, cells grown at 25°C showed vacuolar membrane fluorescence after overnight growth at 37°C whereas the pattern was that of punctate pre-vacuolar compartments (Figure 3D). When GFP–Pep12p was transiently expressed from the GAL1 promoter, and then cells shifted from 25 to 37°C, the pre-existing protein remained on vacuolar membranes for at least 4 h (Figure 3D), eventually disappearing as a result of dilution and/or degradation without ever showing the typical pattern of pre-vacuolar accumulation. Thus, under conditions where delivery is blocked, Pep12p leaves the vacuole very inefficiently, if at all. We conclude that the endosomal location of Pep12p depends primarily on retrieval from PVEs, rather than the vacuole. Snx4p, Snx41p and Snx42p are required for retrieval of Snc1p We next sought to determine whether sorting nexins are involved in other pathways of retrieval from endosomes. We specifically investigated SNAREs that are candidates for direct retrieval from PGEs, namely Tlg1p, Tlg2p and Snc1p. Figure 4A shows that the distribution of all of these was normal in grd19 cells. We tested these proteins in strains lacking each of the other putative sorting nexins (as listed in Figure 3B). The only effects we observed were with Snc1p, which was significantly mislocalized to the vacuole in snx4, ydr425 and ydl113 cells. Because of the similarity of their phenotype to that of snx4, and other data discussed below, we refer henceforth to ydr425w as snx41, and to ydl113c as snx42. In these strains, Snc1p usually was found inside the vacuoles (Figure 4B), indicating entry into the internal membranes of multivesicular bodies, as was observed previously for mislocalized mutant versions of Snc1p (Lewis et al., 2000). Some Snc1p remained on the plasma membrane, indicating that mis-sorting was not complete. This did not seem to be due simply to redundancy of the nexins, because a triple mutant snx4 snx41 snx42 still showed some residual Snc1p at the cell surface (Figure 4B), which is not seen in mutants that completely block the recycling pathway such as ypt6 (Siniossoglou et al., 2000). Figure 4.Specific effects of different sorting nexin mutants. (A) The distribution of GFP-tagged Snc1p, Tlg1p and Tlg2p is the same in wild-type (WT) and grd19 cells. (B) GFP–Snc1p is mislocalized to the interior of the vacuole, identified in the upper panels by double labelling with FM4-64, in snx4, snx41 and snx42 cells. Some residual plasma membrane staining is apparent in some cells (arrowheads), and this is true even in an snx4 snx41 snx42 triple mutant (shown at lower magnification to illustrate the range of phenotypes). (C) Profiles of immunoblots of GFP–Snc1p in the indicated strains, detected with anti-GFP antibody. P indicates the phosphorylated forms, and the proteolytic fragment referred to in the text is also indicated. Note the reduction in phosphorylated forms and increased proteolysis of GFP–Snc1p in snx4 cells. Download figure Download PowerPoint The effect of snx4 on Snc1p could also be observed by immunoblotting. GFP–Snc1p is phosphorylated on the plasma membrane, as revealed by the presence of more slowly migrating forms of the protein, and this has been used as a semi-quantitative assay for plasma membrane localization and hence recycling efficiency (Galan et al., 2001). Figure 4C shows that phosphorylated GFP–Snc1p could be detected in control cells, but not in a ypt6 mutant. It was still detectable in pep12 cells, consistent with continued recycling of Snc1p through PGEs even when delivery to PVEs is blocked. In snx4 cells, the phosphorylated species were reduced significantly, but the most striking effect was a large increase in a proteolytic fragment that is normally only present in minor amounts. This fragment evidently is generated in late endosomes or the vacuole, since it is absent when traffic to these is blocked by deletion of pep12 (Figure 4C). These data confirm that there is substantial mis-sorting of Snc1p in the snx4 mutant. Despite the obvious effects on Snc1p, it was striking that recycling of Tlg1p and Tlg2p was not affected in snx4 cells. We also examined GFP–Chs3p, which is thought to recycle through PGEs, but this too was unaffected (data not shown). However, for none of these proteins has a specific retrieval signal yet been identified. A possible explanation is that there are multiple redundant signals, recognized by different receptors, and that removal of any one is not sufficient to perturb sorting. Interactions between Snx41p, Snx42p and Snx4p Snx41p and Snx42p are related throughout their sequence, and both show a more distant similarity to Snx4p. However, since all three are required for efficient sorting of Snc1p, and do not show an additive phenotype, they are not simply redundant. An alternative possibility is that they are components of a complex. To test this, we co-expressed in yeast functional versions of the proteins tagged with either protein A (PtA) or a peptide epitope (haemagglutinin; HA), isolated the PtA chimeras using IgG–Sepharose beads, and tested for co-isolation of the HA-tagged partner. Using this approach, we were able to co-precipitate Snx41p with Snx4p, and vice versa, and also Snx42p with Snx4p, and vice versa, but could detect no interaction between Snx41p and Snx42p, or between any of these and Grd19p (Figure 5). The efficiency of precipitation of HA-Snx41p was comparable with that of PtA-Snx4p, implying that essentially all of the Snx41p was complexed with Snx4p; the recovery of HA-Snx42p was somewhat less efficient, but still substantial. In contrast, there was little interaction between differently tagged versions of the same protein, although a very weak signal was obtained for HA-Snx4p with PtA-Snx4p. Thus, under the conditions of the experiment, we can detect Snx4–Snx41 dimers and Snx4–Snx42 dimers, but not larger oligomers. From the mutant phenotypes, it appears that both dimers contribute to Snc1p sorting. In agreement with these results, high-throughput two-hybrid screens have also detected interaction of Snx41p and Snx42p with Snx4p, but not with each other (Uetz et al., 2000; Ito et al., 2001). Figure 5.Interactions between nexins. Yeast strains deleted for the appropriate gene or pair of genes were transformed with plasmids expressing the corresponding nexins, one protein A tagged and one HA tagged, as indicated. The protein A-tagged form was isolated, and co-purifying HA-tagged protein detected by immunoblotting (arrowheads). The immunoprecipitates (IP) contain more cell equivalents than the extracts (Ex), but the ratio is the same in each case. The band indicated with an asterisk represents binding of the anti-HA antibody to Snx42p– protein A. Download figure Download PowerPoint Interaction of Snx4p with Snc1p To explore the interactions between the Snx4p family and Snc1p, we co-expressed a PtA-tagged version of Snc1p with HA-tagged Snx4p, exposed isolated spheroplasts to the reversible cross-linker dithiobis(succinimidyl propionate) (DSP) and isolated the Snc1p on IgG–Sepharose. HA-Snx4p did not co-precipitate significantly with Snc1p in the absence of cross-linker, but was clearly detectable after cross-linking (Figure 6A). Controls showed that PtA fused to dihydrofolate reductase could not be cross-linked to Snx4p, and there was also no detectable cross-linking of PtA-Snc1p to HA-Grd19p (Figure 6A). Figure 6.Cross-linking of Snx4p to Snc1p. (A) Yeast strains deleted for snx4 or grd19 were transformed with plasmids expressing HA-tagged Snx4p or Grd19p, together with a plasmid expressing protein A–Snc1p or, as a control, protein A–dihydrofolate reductase. Spheroplasts were treated with the reversible cross-linker DSP prior to lysis, the protein A fusions immunoprecipitated with IgG–Sepharose beads, and protein A-tagged and associated HA-tagged proteins detected by immunoblotting. Results are shown for the cell extracts (Ex) and precipitates (IP). The band indicated by an asterisk corresponds not to HA-Snx4p, but to a small amount of IgG heavy chain eluted from the beads, which weakly cross-reacts with the secondary antibodies used in this experiment. (B) The effects of the W86R mutation in Snc1p on Snx4p binding were tested as in (A), except that in this experiment the bound protein was eluted from the IgG–Sepharose beads by specific proteolytic cleavage between the protein A and the Snc1p. Download figure Download PowerPoint Previous studies identified one point mutation in the cytoplasmic domain of Snc1p that caused its mistargeting to the vacuole, namely W86R (Lewis et al., 2000). As shown in Figure 6B, this mutation also substantially reduced cross-linking of Snc1p to Snx4p, providing further evidence for the specificity and functional relevance of the interaction. We also failed to see significant cross-linking of Snx4p to the Pep12p derivative that is found in PGEs (data not shown). Thus, our data indicate a specific association of Snx4p with Snc1p, suggesting a direct role for this sorting nexin in the recycling of Snc1p. Snx4 acts independently of retromer The data suggest that Snx4p and its partners act in PGEs, and are quite independent of retromer, which is thought to act in PVEs. In agreement with this, we have shown previously that retromer mutants do not affect the cycling of Snc1p (Lewis et al., 2000). Furthermore, Figure 7A shows that the only sorting nexin mutants that substantially mis-sort carboxypeptidase Y are the retromer components, and to a lesser extent mvp1, whereas snx4 and the others do not. Figure 7.The Snx4 family of nexins have functions distinct from that of retromer. (A) Detection of carboxypeptidase Y secreted by each sorting nexin mutant. Note that only vps5, vps17 (both encoding retromer subunits) and mvp1 show a detectable phenotype. (B) Synthetic growth defect. The indicated mutants, in two strain backgrounds, were grown to saturation in liquid culture and then spotted at high and low concentrations on plates. Download figure Download PowerPoint That retromer and Snx4p have distinct functions is also indicated by a genetic interaction between them. We observed that in one genetic background (strain SEY6210), there was a synthetic growth defect between deletions of snx4 and the retromer component vps17: the double mutant grew significantly more slowly than either single mutant (Figure 7B), though in liquid culture the cells did eventually reach comparable densities. Growth could be restored to the double mutant by transformation with a plasmid encoding either of the genes (data not shown). This demonstrates formally that Snx4p and Vps17p can each function in the absence of the other, since each can improve growth of the double mutant. PI3P-dependent binding of Snx4p, Snx41p and Snx42p to membranes PX domains bind to PI3P, which is found in endosomes and the vacuole. To localize Snx4p and its partners, we tagged each with GFP and examined them in live cells. When expressed at endogenous levels, GFP–Snx4p and GFP–Snx42p were visible, though very