In this paper we have used a GFP-tagged version of the exocytic SNARE Snc1p to probe the recycling pathway from the plasma membrane and the sorting events that are involved. Because the fusion protein is functional, we assume that its behavior broadly reflects that of its untagged counterpart. However, our conclusions are derived from the behavior of the chimera and do not require its transport kinetics to be identical to those of the wild-type protein.
It is generally assumed that SNARE proteins that travel on vesicles are recycled for reuse, and it is clear that GFP-Snc1p is endocytosed and reenters exocytic vesicles. Because mutations that block traffic through the Golgi lead to rapid removal of GFP-Snc1p from the cell surface, it seems likely that recycling occurs via the Golgi rather than by a direct endosome–plasma membrane route. It is difficult to exclude completely the possibility that Golgi function is required only indirectly for recycling, as it is to some extent for the later stages of the endocytic pathway (Hicke et al., 1997
), but if internalized Snc1p is to be used for subsequent rounds of exocytic traffic, it must return to the Golgi. Independent evidence for such a route is provided by our observation that FM4-64 dye is efficiently transferred to Golgi structures, as defined by the presence of Sec7p and their morphological alteration in a sec14
mutant. One consequence of recycling via the Golgi is that redelivery to the surface occurs in a polarized manner, a feature that may be important for the specific targeting of some recycling proteins such as the chitin synthase subunit Chs3p (Holthuis et al., 1998b
GFP-Snc1p recycling is independent of the late endosome/prevacuolar compartment that has been characterized previously. Thus, although it requires the presence of the syntaxins Tlg1p and Tlg2p, it does not require the late endosomal SNARE Pep12p. Furthermore, it is unaffected by vps4
, a mutation that inhibits exit from the prevacuolar compartment, or by removal of Vps5p, Vps17p, and Vps35p, which are components of the retromer coat that mediates the retrieval of proteins from this compartment (Seaman et al., 1998
). There must therefore be two distinct routes from the endocytic pathway to the Golgi complex, one from early endosomes and one from later ones. Given the involvement of Tlg1p and Tlg2p in Chs3p trafficking (Holthuis et al., 1998b
), it is likely that Chs3p follows a cycling itinerary similar to that of Snc1p.
The choice that faces an endocytosed protein is best illustrated by comparing the fate of GFP-Snc1p with that of a mutant version in which the TMD has been exchanged for that of Sso1p. Both versions are endocytosed in an END4
-dependent manner, and both require an endocytosis signal that is similar to that on the animal cell versions of Snc1p (Grote et al., 1995
). This in itself is striking, because the requirements for endocytosis seem to differ somewhat in yeast and animal cells.
Once internalized, the altered form of GFP-Snc1p proceeds to the vacuole, apparently by default. In contrast, the version with its normal TMD is transferred to the Golgi in a step that is crucially dependent on sequences within and adjacent to the TMD. This sorting event is likely to occur in a compartment marked by Tlg1p and Tlg2p, for several reasons. First, the Tlg proteins are required for recycling, whereas Pep12p is not. Second, although Tlg1p is not present on the plasma membrane Snc1p can readily be found in complexes containing Tlg1p (Holthuis et al., 1998a
), suggesting that fusion occurs between endocytic vesicles containing Snc1p and a Tlg1p-bearing membrane. Third and most importantly, subtle alterations to the Snc1p TMD such as the L96V mutation result in the accumulation of the protein in Tlg1p-positive structures. Because Snc1p can be chased reversibly from these structures to the cell surface when endocytosis is blocked by the cmd1-1
mutation, they evidently comprise a station on the recycling pathway. We interpret the accumulation of the mutants there as being due to the slowing of their retrieval when recognition of the sorting signal is impaired, which would imply that these Tlg1p-positive structures are where sorting occurs. This conclusion fits well with our previous finding that Tlg1p and Tlg2p are sufficient to mediate recycling of Chs3p even in the absence of Pep12p and Vam3p, and that in wild-type cells the internal pool of Chs3p cofractionates with Tlg1p and Tlg2p (Holthuis et al., 1998b
The sensitivity of GFP-Snc1p retrieval to mutations within the TMD suggests that this domain is recognized by a transmembrane receptor and actively recruited into carriers destined for the Golgi. It also provides strong evidence that the endosomes in which sorting occurs are physically distinct from the late Golgi compartment (the trans-Golgi network [TGN] equivalent). This is because the TMD requirements for transport from Golgi to cell surface are much less specific than the requirements for retrieval. If endocytosis occurred directly to the TGN, then Snc1p chimeras with heterologous TMDs would simply return to the surface, rather than pass to the vacuole.
Figure illustrates a simple model for the recycling pathway. In this model, late Golgi (TGN) membranes segregate into exocytic vesicles (containing Snc1p) and others containing the resident TGN proteins, including Tlg1p and Tlg2p, which do not progress to the plasma membrane. The Tlg1p detected in small buds by immuno-EM may be in these nonexocytic carriers, because only small amounts of Tlg1p are found on the cell surface even when endocytosis is blocked (Holthuis et al., 1998b
; our unpublished observations). Late Golgi-derived vesicles subsequently fuse with Snc1p-bearing endocytic vesicles to form the earliest endosomes, which may continue to receive vesicles from both sources. From these structures most TGN proteins, and others from the plasma membrane such as Snc1p and Chs3p, are selectively returned to the Golgi. The remaining proteins reach a later endosome marked by Pep12p. Subsequent steps involve retromer-dependent removal of proteins such as the carboxypeptidase Y (CPY) receptor Vps10p in vesicles bound for the Golgi and budding of membranes into the lumen of the endosome to form a multivesicular body, which eventually fuses with the vacuole (Odorizzi et al., 1998
; Seaman et al., 1998
Figure 10 Proposed outline model for endocytic traffic. Bold names indicate major locations of the indicated proteins; thin arrows represent vesicular transport steps; thick arrows show “default” pathways, which may correspond to compartment maturation (more ...)
Recruitment of Snc1p and other recycling proteins into Golgi-bound vesicles is likely to be mediated ultimately by a cytoplasmic coat. We have shown that the retromer coat is not required for retrieval of GFP-Snc1p. Also, retrieval is not affected by deletion of any of the known adaptin homologues in yeast (APL1-6
, and APS1-3
; Van Horssen and Pelham, unpublished observations), although this does not rule out the possibility that clathrin is involved. The COPI coat is a good candidate, because retrieval appears more sensitive to mutations in coatomer subunits than is secretion itself. This would fit with results in animal cells that implicate COPI in endosomal sorting (see Daro et al., 1997
, and references therein). However, the requirement for Golgi function for Snc1p recycling and the known involvement of COPI in this makes it difficult to draw a firm conclusion.
Our model predicts that organelles containing Tlg1p and other TGN proteins are physically and functionally heterogeneous. There is indeed evidence for such heterogeneity: the proteins are found in membranes of two different densities, which we have previously suggested might correspond to Golgi and early endosomes (Holthuis et al., 1998a
), and immunofluorescence shows that only a subset of the structures containing Tlg1p or Tlg2p carry the Golgi marker Sec7p. A more specific prediction is that the earliest endocytic structures should contain markers destined both for recycling and transport to the vacuole, but this has proved hard to demonstrate: FM4-64 appears to be rapidly transferred to the Golgi, and a GFP-tagged version of the alpha factor receptor Ste2p rapidly reaches Pep12p-containing endosomes after uptake is stimulated with alpha factor (our unpublished observations; also see Holthuis et al., 1998b
). We note that passage through Tlg1p-containing endosomes may not always be obligatory for endocytosed material and vacuolar hydrolases. Even in the absence of both Tlg1p and Tlg2p, FM4-64 can reach endosomes, probably by direct fusion of primary endocytic vesicles with membranes bearing Pep12p, and a substantial proportion of newly synthesized CPY can reach the vacuole (Holthuis et al. 1998a
How proteins present in Tlg1p-containing endosomes are transferred to later ones marked by Pep12p is an interesting question. This appears to be the default pathway once retrieval signals are removed, which argues against a highly selective mechanism. The structures containing both Tlg1p and Pep12p that we observe by immuno-EM are plausible intermediates in the process and could in principle arise by fusion of the two types of endosome (or of membranes derived from them). However, there is another possibility. The later stages of endocytosis seem to occur by maturation and fusion of membranes to the vacuole, and this requires Pep12p to be removed, because it does not accumulate on the vacuole. Furthermore, vps mutations that block recycling of Vps10p to the Golgi also trap Pep12p in prevacuolar structures. Thus, it may be that Pep12p recycles through the Golgi (Figure , route 2) and is delivered to early endosomes, promoting their maturation into later structures and becoming concentrated as other proteins are removed.
The existence of two distinct pathways back from endosomes to the Golgi explains several previous observations. For example, although Snc1p, Chs3p, Tlg1p, and Tlg2p seem to recycle mainly from early endosomes, there is good evidence that the late Golgi proteins Kex2p and DPAPA contain cytoplasmic signals that mediate their retromer-dependent retrieval from later endosomes, and that they can, like the CPY receptor Vps10p, reach the class E compartment (an abnormal prevacuolar structure) in appropriate vps
mutants (Voos and Stevens, 1998
; Nothwehr et al., 1999
). However, both DPAPA and Kex2p have a second signal, which slows their entry into this compartment and which has been interpreted as a TGN retention signal (Brickner and Fuller, 1997
; Bryant et al., 1997
). We suggest that these are in fact signals for retrieval from early endosomes, and that these proteins can follow both routes. The model also explains why, although GFP-Snc1p sorting is severely disrupted in a tlg2
mutant, CPY sorting is barely affected and no more than half of the DPAPA protein is lost to the vacuole: in this mutant, the late endosome retrieval pathway should remain functional (Abeliovich et al., 1998
; Holthuis et al., 1998a
; Nichols et al., 1998
; Seron et al., 1998
The precise roles of Tlg1p and Tlg2p in these retrieval pathways remain to be worked out. Both are required for the route from early endosomes, but their mutant phenotypes are different—the pattern of GFP-Snc1p is qualitatively different in tlg1
cells. As discussed previously (Holthuis et al., 1998b
; Nichols et al., 1998
), Tlg1p has the unusual property of binding to other syntaxins (Tlg2p and Sed5p) and thus could in part help target vesicles (on either route) to the Golgi. Tlg2p is a more typical syntaxin and might serve as a vesicle acceptor in early endosomes, the late Golgi, or both. However, because removal of either can potentially alter the location of the other, it is very difficult to discern their individual functions from these genetic experiments.
The two routes from endosomes to the Golgi use different machinery and most likely originate in distinct organelles, but whether they have different endpoints is less clear. Returning Golgi proteins following route 1 in Figure might be delivered selectively to the late Golgi, perhaps using Tlg2p. However, the relatively efficient sorting of CPY in tlg mutants argues that the Vps10p recycling pathway (route 2) can use Sed5p. Other possibilities are that both routes use early and late Golgi interchangeably, or that all traffic to the Golgi uses Sed5p.
The concept of traffic from endosomes to the early Golgi may help explain the recent finding that in a different yeast strain, W303, Tlg1p is essential for transport of CPY from the ER to the Golgi. In these cells the distribution of Tlg1p is reported to overlap substantially with that of Sed5p (Coe et al., 1999
). Our immuno-EM studies confirm that Tlg1p is capable of reaching membranes that contain Sed5p, although we found double-labeled structures to be infrequent. Together, the evidence suggests that Tlg1p helps vesicles derived from the endocytic pathway fuse with the Sed5p compartment, and that delivery of some component via this route is necessary for normal Golgi function. Evidently in the W303 strain this route is more dependent on Tlg1p, or more important for Golgi function, than in the SEY6210 strain that we have used. Why this should be is not obvious, but W303 cells are also more sensitive to disruption of YPT6
, a gene whose mutant phenotype is strikingly similar to that of tlg1
, and this has been shown to be due to mutation of the SSD1
locus in W303 (Li and Warner, 1996
; Tsukada and Gallwitz, 1996
). The differences in Tlg1p distribution presumably reflect different rate-limiting steps in its itinerary and illustrate the limitations of using circulating integral membrane proteins as compartment markers. Peripheral proteins that dissociate when a compartment matures or fragments may be more useful, and we are currently seeking early endosomal markers of this type.
The protein sorting events in the prevacuolar compartment in yeast are relatively well characterized, because defects at this point have an easily scored vps phenotype, and many mutants have been isolated. In contrast, sorting defects in early endosomes have much more subtle phenotypes, and this process has been harder to detect and study. The convenience of GFP-Snc1p as a marker should facilitate future studies, including the identification of the receptor and other machinery responsible for its retrieval.