Since the identification of AP-3 in animal cells, much effort has been directed at determining the function of this novel adaptor-related complex. This complex does not appear to colocalize or copurify with clathrin and, therefore, may function independently of clathrin coats (Newman et al., 1995
; Simpson et al., 1996
; Dell'Angelica et al., 1997b
; Simpson et al., 1997
). Cytological studies have suggested that the mammalian AP-3 complex is involved in transport from the TGN or functions in an endosomal compartment (Simpson et al., 1996
; Dell'Angelica et al., 1997a
; Simpson et al., 1997
). Further information on the function of AP-3 came from the finding that the garnet
gene of Drosophila
encodes a protein closely related to the δ subunit of AP-3 (Ooi et al., 1997
; Simpson et al., 1997
). Mutations in garnet
lead to reduced pigmentation of the eyes and other tissues. Pigment cells are present, but these have few or no pigment granules, which are thought to be lysosome-like organelles. Therefore, it was suggested that AP-3 might be involved in sorting to or biogenesis of lysosomal organelles. Our studies showing a requirement of AP-3 for processing and sorting of ALP support this idea, and provide direct functional evidence that the AP-3 complex is required for sorting to the vacuole, a lysosome-like compartment. Interestingly, the pathway that AP-3 mutants block is not the well studied Vps pathway, but the alternate route. This is consistent with the hypothesis that the garnet
mutations affect formation of a specialized lysosome-like organelle, which might have different trafficking requirements for biogenesis than normal lysosomes.
Where the yeast AP-3 complex functions in the alternative pathway from the late Golgi to the vacuole still needs to be defined. Presumably, the AP-3 complex serves as a coat protein; binding to cytoplasmic tails, sequestering cargo proteins, and directing vesicle formation. It is possible that the AP-3 complex binds to the cytoplasmic face of the late Golgi/TGN, directing selective cargo (e.g., ALP) away from the secretory and Vps pathways. The pALP that accumulated in the apm3-Δ mutant sedimented at 100,000 g where Golgi, endosomes, and small vesicles are found. However, immunofluorescence staining indicated that ALP localized to structures distinct from the late Golgi protein Kex2p, which showed a normal localization pattern. Also, ER and Golgi modifications of ALP occurred with normal kinetics in AP-3 mutants (Fig. A), suggesting that both early and late compartments of the Golgi are unaffected in the absence of the adaptor. Nonetheless, the AP-3 complex could function at the late Golgi/ TGN and ALP becomes trapped in a subcompartment budding from this organelle.
We also found that substantial ALP was diverted to the Vps pathway in the AP-3 mutants, but its processing was very slow. Therefore, the ALP-staining structures may represent a normal subcompartment along the Vps pathway and this was visualized in the apm3 mutant because ALP's transit through this compartment was delayed. Independent studies have indeed shown that when the sorting information in the cytoplasmic tail of ALP is mutated, it traffics through the Vps pathway, resulting in a kinetic delay in the mutant protein's arrival at the vacuole (Piper et al., 1997). However, the mutant ALP did not appear to localize with the punctate staining pattern we observed in the AP-3 mutant.
This raises an alternative possibility: ALP is able to enter its normal trafficking pathway but becomes trapped in an intermediate compartment in the adaptor mutant. In this case, AP-3 might not be involved directly in cargo selection at the late Golgi, but could function at some later compartment, such as another endosomal/prevacuole intermediate. Over time, sorting receptors or other membrane components of the sorting machinery might not be recycled to the Golgi, resulting in ALP slowly leaking into the Vps pathway. The punctate staining pattern suggests that the ALP might be trapped in vesicles that cannot fuse with their target membrane, such as an endosomal compartment or the vacuole itself, although by EM there was no dramatic accumulation of vesicles like that seen with class D vps mutants. If such a fusion defect existed, it might be caused by reduced recycling of some component(s) (e.g., a vesicle-soluble-N-ethylmaleimide-sensitive factor attachment protein receptor [v-SNARE]) to the Golgi that would need to be included in transport vesicles for fusion at a later step.
An endosomal function for AP-3 is also consistent with the findings that AP-3 mutants partially suppress the growth and Ste3p internalization defects caused by yeast PM casein kinase 1 deficiency (Panek et al., 1997
). If AP-3 functions at an endosomal compartment where the endocytic and Golgi to vacuole pathways overlap, loss of function of the complex might affect both the ALP pathway and a recycling pathway to the cell surface, resulting in the suppression of the kinase endocytic defect. The genetic interactions of apm3-Δ
also suggest a possible functional overlap of an endocytic route and the ALP sorting pathway. Alternative possibilities are that AP-3 has more than one sorting function in the cell or that some of the proteins that normally traverse the ALP pathway are diverted to secretory vesicles and traffic to the cell surface in AP-3 mutants, where they might somehow suppress the yck
endocytic defect. Identification of the kinase substrates and localization of the AP-3 complex within the cell should help clarify the connections of the AP-3 complex with both Golgi to vacuole transport and endocytosis.
Although it is not clear at what step the AP-3 complex functions in sorting ALP to the vacuole, existing mutations seem to define at least three distinct steps along this pathway (Fig. ). The late acting Vps proteins, including Vps33p, a Sec1p homologue (Banta et al., 1990
; Wada et al., 1990
), Vam3p, a syntaxin-related target (t)-SNARE of the vacuole membrane (Darsow et al., 1997
; Nichols et al., 1997
; Wada et al., 1997
), and Ypt7p, a Rab homologue (Wichmann et al., 1992
; Haas et al., 1995
; Wada et al., 1996
), are thought to function in fusion of endosome- derived membranes or vesicles directly with the vacuolar membrane. These gene products appear to be required for the final stages of transport in both the Vps and ALP pathways. Vps41p/Vam2p (Cowles et al., 1997
; Nakamura et al., 1997
) and Vps39p/Vam6p, which were recently shown to physically interact (Nakamura et al., 1997
), may also act at a distinct, possibly later, step than AP-3. Null mutants of these genes show a block in both CPY sorting and ALP processing and also accumulate fragmented vacuoles containing the V-ATPase and ALP (Raymond et al., 1992
; Cowles et al., 1997
; Nakamura et al., 1997
were also identified in a screen for cvt
mutants, which are defective in the cytoplasm to vacuole transport pathway (Harding et al., 1995
). However, recent studies using a vps41-ts
allele suggested that the primary defect resulting from loss of Vps41p function is in the ALP pathway, since only after prolonged periods at the nonpermissive temperature did the vps
sorting defect surface (Cowles et al., 1997
). These phenotypes are quite distinct from those found in AP-3 null mutants which exhibit no obvious vps
phenotypes, localize the V-ATPase to the vacuole normally and show no extensive elaboration of any abnormal membrane organelles. AP-3 is also not required for the Cvt pathway (Huang, K., and S. Lemmon, unpublished observations). Therefore, the AP-3 complex most likely acts at a different step than the Vps41p(Vam2p)– Vps39p(Vam6p) complex.
Figure 9 Models for the Vps-dependent and alternative ALP pathways to the vacuole. (A) Proteins such as the Vps10p CPY sorting receptor, Kex2p and the vacuolar ATPase membrane protein, Vph1p, exit the late Golgi in transport vesicles, which then fuse with (more ...)
In our attempt to further characterize the pathway responsible for the residual ALP processing in AP-3 mutant cells we obtained an astonishing result. In the context of a vps
class E mutant, loss of AP-3 function resulted in reappearance of the V-ATPase on the vacuolar membrane. Strictly speaking, this was not suppression of the class E phenotype(s) since Kex2p did not return to the Golgi, but was also transported to the vacuole. In addition, p2CPY was secreted from the cell (data not shown), indicating these cells retained their vps
phenotype. Interestingly, ALP localization was still predominantly in small vesicular structures throughout the cytosol. One model to explain these observations is that a regulatory protein(s) normally sorted into the ALP–AP-3–dependent pathway becomes diverted through the Vps pathway in the AP-3 mutant. This might now enable fusion of the aberrant class E compartment with the vacuole or permit prevacuole–prevacuole fusion, which could mature into a large vacuolar-like organelle. A candidate for such a protein is Vam3p, which has recently been suggested to traffic through the ALP– AP-3 pathway (Piper et al., 1997). In a normal cell it would be highly desirable for a t-SNARE, such as Vam3p, to bypass at least some compartments of the Vps pathway to prevent premature interaction with its v-SNARE partner, perhaps the newly identified Nyv1p (Nichols et al., 1997
). This sort of quality control system would be essential for maintenance of an endosomal/prevacuole compartment, from which proteins need to recycle to the Golgi and through which endocytic traffic must pass.
This model could also explain why the genetic interactions we observed were largely restricted to the combination of apm3-Δ
with class D vps
mutants, which are blocked before fusion with the prevacuole. Mutants which can form a functional prevacuole or acidifying compartment like the vacuole (such as class E, B, and A vps
mutants; Raymond et al., 1992
) would be able to survive quite well even in the absence of AP-3 function. There is also evidence that some class D mutants have endocytic defects (pep7
; Webb et al., 1997
, and vps45
; our unpublished observations), suggesting that some components involved in vesicle fusion with the prevacuole may be shared by the endocytic and Golgi to vacuole pathways. In the absence of AP-3 and certain class D genes all three pathways to the vacuole would be blocked.
The model in Fig. shows the requirement of the AP-3 complex for transport of ALP from the late Golgi to the vacuole. At the present time, it is not clear how many intermediate steps there are along the ALP–AP-3 pathway. It is possible that there is a single vesicle intermediate for transport of ALP from the late Golgi directly to the vacuole, or that ALP traffics through a Vps-independent endosomal compartment. Although the model shown suggests AP-3 acts at the level of the late Golgi, as discussed above, it could act at such an endosomal intermediate (e.g., Fig. B
). A more speculative model is considered in Fig. B
in which Golgi-derived vesicles that follow the Vps pathway initially fuse with an early endosome, and that the ALP- (and Vam3p-) containing vesicles fuse with the late endosome. The Vps pathway would then converge with the ALP pathway at the late endosome/prevacuole. This idea allows for maintenance of an early endosome recycling compartment and is consistent with the observation that Vam3p, Vps33p, Ypt7p, and presumably other late acting Vps proteins are required for a late stage of transport to the vacuole of both ALP and proteins of the Vps pathway. It has been proposed that the class E compartment is an abnormal late endosome/prevacuolar compartment, since it appears as one or two bright spots next to the vacuole (Raymond et al., 1992
; Piper et al., 1995
; Rieder et al., 1996
; Babst et al., 1997
), and by EM it appears as stacks of curved membrane cisternae (Rieder et al., 1996
; Babst et al., 1997
). However, recent studies indicate that the tubular structures of the aberrant class E organelle are more reminiscent of early endosomes observed in yeast (Prescianotto-Baschong and Riezman, 1998
). Moreover, morphological analysis of an end13
is an allele of VPS4
[Munn and Riezman, 1994
]) indicates endocytosis is retarded at an early, postinternalization endocytic step (Riezman, H., personal communication). As more gene products involved in the alternative ALP pathway to the vacuole are identified, it should be possible to resolve these two models.