In this paper, we have used immunoelectron microscopy to examine the localization in Drosophila
oocytes of the exocyst component Sec5. We find that the plasma membrane localization of Sec5 seen by light microscopy reflects a substantial amount of the protein being present in clathrin-coated pits and vesicles. This was unexpected as the exocyst had previously been shown to be required for fusion of Golgi-derived exocytic vesicles to the plasma membrane. It should be stressed that our observations do not question this role for the complex. In addition to the Sec5 present in coated structures, we could clearly detect Sec5 in uncoated areas of the plasma membrane, albeit at a lower linear density. Moreover, germline clones of a null allele of Sec5, sec5E10
, and of null mutants in other exocyst components, Sec6 and Sec8, arrest very early in germline cyst development with multinucleate cells containing ring canals detached from the cell surface, presumably reflecting a severe defect in the delivery of new plasma membrane that has prevented germ cell growth and division (Murthy and Schwarz, 2004
; Murthy et al., 2005
; unpublished data). However, our observations raise the possibility that Sec5, and potentially other exocyst components, not only acts in traffic from the Golgi to the plasma membrane but is also recruited to coated pits to act at some step in the membrane traffic events after endocytic internalization.
The primary cargo for clathrin-dependent endocytosis in the oocyte is the receptor Yolkless, which mediates the uptake of massive amounts of yolk during the 12-h period of vitellogenesis during developmental stages 8–10 (DiMario and Mahowald, 1987
). Because Yolkless does not accumulate in the yolk granules, it must recycle many times. The oocyte endocytic pathway has been examined by using the shibire
temperature-sensitive mutant in dynamin to reversibly block budding of clathrin-coated vesicles (Tsuruhara et al., 1990
). This examination has shown that the network of tubes and small yolk-containing endosomes under the surface is lost in <10 min after a block in endocytosis and that these structures reform over 3–5 min when endocytosis is restored. Thus, it appears that endocytosed vesicles fuse with each other to continuously create early endosomes. Yolk condenses in these endosomes, and they emanate tubes that have been suggested to recycle the receptor back to the surface, whereas the residual yolk-containing core fuses with larger yolk granules deeper inside the oocyte (; Roth and Porter, 1964
; Giorgi and Jacob, 1977
; Tsuruhara et al., 1990
). Thus, at least two membrane fusion events must occur to sustain the endocytic cycle of Yolkless—fusion of the endocytosed vesicles to other vesicles or nascent endosomes (, step 1), and then fusion of the recycling tubular carriers back to the plasma membrane (, step 2). The recruitment of Sec5 to the forming coated pits could allow it to participate in one or both of these fusion events.
Figure 9. Schematic illustration of the recycling route of Yolkless. Yolkless binds yolk and both are endocytosed in clathrin-coated vesicles. Yolk condenses in early endosomes and is excluded from tubules that are thought to recycle Yolkless (dotted line). The (more ...)
Further suggestion that Sec5 acts in the endocytic cycling of Yolkless comes from the sec5E13
allele that expresses a truncated form of Sec5 lacking the COOH-terminal 534 of 894 residues. Germline clones for sec5E13
still develop oocytes, but these show a defect in yolk uptake (Murthy and Schwarz, 2004
). In the initial characterization of this sec5E13
allele it was noted that Yolkless accumulated in cytoplasmic structures, which were suggested to reflect trapping of the protein in the secretory pathway due to a block in exocytosis (Murthy and Schwarz, 2004
). However, by immunoelectron microscopy and double label immunofluorescence it appears that the bulk of Yolkless accumulates in late endosomal compartments.
We suggest that the sec5E13 allele might be defective in the fusion event mediated by the pool of Sec5 present in clathrin-coated pits. If the defective step is the fusion of uncoated vesicles with each other or with early endosomes, then the vesicles may fuse aberrantly with later endosomes. If these lacked the machinery for Yolkless recycling, the protein would be trapped. Alternatively, the sec5E13 allele could cause a defect in the fusion to the plasma membrane of the carriers that recycle Yolkless from endosomes back to the surface. Although there is not a massive accumulation of uncoated vesicles near the surface of the sort seen in yeast when exocyst function is inactivated, it may be that even a small amount of accumulation might sequester machinery for this transport step. Thus, the machinery for formation or consumption of endosome to plasma membrane carriers would be depleted from early endosomes, and hence Yolkless would be trapped and instead be delivered with the yolk to later endosomal compartments. In either case, the presence of Sec5 in clathrin-coated pits could be a means to ensure that it is ready to act as soon as vesicles uncoat, or postendosomal carriers are formed, so that the recycling of Yolkless can proceed as efficiently as possible.
It should be stressed that the aforementioned model is not proven, and alternative interpretations for the phenotype of the sec5E13
allele are possible. One alternative is that the initial delivery of newly synthesized Yolkless from the Golgi to the plasma membrane is defective. However, there does not appear to be a general block in Golgi to plasma membrane traffic in the sec5E13
allele. First, the plasma membranes of sec5E13
oocytes and nurse cells are able to expand to a near normal size (Murthy and Schwarz, 2004
). In null alleles for Sec5 and other exocyst subunits, the germline cyst develops to a diameter of only 20–40 μm divided into two to three multinucleate cells, whereas by stage 10 of oogenesis the sec5E13
egg chamber has developed to a similar size to wild type (Murthy and Schwarz, 2004
; Murthy et al., 2005
). This development requires formation of 15 nurse cells, each of ~60 μm diameter, and an oocyte of ~150 μm diameter. Thus, the sec5E13
allele is able to sustain a very large increase in plasma membrane expansion beyond that seen when exocyst function is lost. This result is also in contrast to mutants with defects in other components of the exocytic machinery such as syntaxin-1A
, and rop
Sec1), which fail to develop oocytes or give miniature eggs, consistent with secretion being necessary for growth of the oocyte (Ruden et al., 2000
). Moreover, we find that two plasma membrane proteins, syntaxin-1A and E-cadherin, are still delivered to the plasma membrane in sec5E13
germline cells and do not accumulate in internal structures. Thus it seems that in the sec5E13
allele the majority of traffic from Golgi to plasma membrane is normal. However, we cannot exclude the possibility that there are two routes from the Golgi to the plasma membrane with a subset of proteins, including Yolkless, being delivered to the surface via recycling endosomes, as has been suggested to occur in mammalian cells (Ang et al., 2004
). In this case, the Yolkless-containing carriers in the sec5E13
allele might be unable to fuse with the early endosome and instead fuse with later endocytic structures from which Yolkless cannot escape.
The long time scale of vitellogenesis and the uncertainty about the precise steps involved means that it is not possible at present to be certain what step in the trafficking of Yolkless is defective in the sec5E13
allele. However, a specific defect in the postinternalization recycling route, rather than Golgi to plasma membrane transport, would correlate well with the presence of Sec5 in clathrin-coated pits and vesicles. Indeed, it is conceivable that the exocyst remains attached to membranes throughout the endocytic cycle. In mammalian cells, components of the exocyst have been localized to recycling endosomes and shown to be effectors for two small GTPases, Arf6 and Rab11 (Prigent et al., 2003
; Zhang et al., 2004
). Moreover, Exo70 was found to be recruited to a perinuclear compartment, possibly the recycling endosome, by the μ subunit of the clathrin adaptor complex AP1B (Folsch et al., 2003
). We tested several possible interactions that might explain the recruitment of Sec5 to coated pits, but these proved negative. Thus a GST fusion to the cytoplasmic tail of Yolkless did not extract Sec5 or the endocytic adaptor Numb from ovary lysates. In addition, immunoprecipitated Sec5 did not coprecipitate Numb or the clathrin adaptor complex AP2 (unpublished data).
The COOH-terminal part of Sec5 that is missing in sec5E13
is well conserved in evolution (30% identical between humans and Drosophila
vs. 44% for the part that remains). The suggestion that this region of Sec5 plays a role in exocyst function distinct from fusion of Golgi-derived vesicles is supported by the observation that an equivalent truncation of the yeast protein does not perturb growth, in contrast to deletion of the whole protein, which is lethal (TerBush et al., 1996
). Although endocytic recycling also continues in these mutant yeast, it is temperature sensitive. At the nonpermissive temperature, endocytosed a
-factor receptor accumulates in a cytoplasmic haze rather than endosomes, suggesting that a vesicle fusion step is blocked. It may be that at the permissive temperature other components of the exocyst are sufficient to sustain the endocytic function of the complex that involves the COOH terminus of Sec5. Interestingly, the yeast protein Rcy1p has been shown to act in recycling of endocytosed proteins and lipid markers (Wiederkehr et al., 2000
; Galan et al., 2001
), and this protein is related to the COOH-terminal part of the exocyst subunit Sec10. Of the other quatrefoil complexes in the cell, GARP/VFT has only four subunits, suggesting that this number may be sufficient for action at a single SNARE-dependent membrane traffic step (Conibear et al., 2003
). The COG complex has eight subunits like the exocyst, but it appears to act in at least two steps in the Golgi apparatus (Wuestehube et al., 1996
; Whyte and Munro, 2001
; Ram et al., 2002
). The central role that the exocyst plays in exocytosis means that deletion of subunits leads to a loss of cell viability, which may have masked other roles for the complex. Indeed, it has been reported recently that the requirement for a subset of exocyst subunits, including Sec5p, for growth of yeast can be by-passed by overexpressing the small GTPase Sec4p or the SNARE-binding protein Sec1p (Wiederkehr et al., 2004
). This finding suggests that in yeast Sec5p is not obligatory for Golgi to plasma membrane transport. Moreover, a recent genome-wide RNAi screen in Caenorhabditis elegans
identified Sec5, but not other exocyst components, as one of several genes whose knockdown causes defects in yolk granule formation in oocytes (Sonnichsen et al., 2005
). In addition, a hairpin RNAi construct against Drosophila
Sec10 was found to only show phenotypes in a small subset of tissues, implying that this subunit may be less critical for exocytosis (Andrews et al., 2002
). We suggest that the exocyst acts in more than one membrane traffic step and that the sec5E13
allele uncouples the exocytic function of Sec5 from a role in the endocytic recycling pathway. Therefore, it may prove fruitful to look in more detail at the role the exocyst plays in postinternalization membrane traffic.