In this study, we show that the ear domain of the AP-3 δ subunit, Apl5, binds to Vps41 in context of the HOPS holocomplex and present evidence that consumption of post-Golgi AP-3 vesicles at the vacuole requires Vps41/HOPS and other vacuole docking and fusion factors. We propose that AP-3 remains on the vesicle at least until docking or fusion, and that the major function of HOPS within the AP-3 trafficking pathway is to tether AP-3 vesicles to the vacuole. A working model based on these observations is shown in .
Working model for the AP-3 pathway in budding yeast. See text for discussion.
Purified Vps41 binds Apl5-ear (), indicating that Vps41 directly mediates the primary interaction between Apl5 and HOPS. However, efficient binding of Vps41 to the Apl5-ear requires the HOPS-specific subunit Vps39, but not the Vps-C core subunit Vps16 (). Vps16 is still required for efficient binding of the remaining HOPS subunits to Apl5-ear (B). These results are perhaps best explained by the subunit arrangement of the HOPS complex (D and ). Vps39 associates with Vps41 directly, whereas Vps16 does not appear to directly interact with Vps41 (Rieder and Emr, 1997
; Wurmser et al., 2000
; our unpublished results). The absence of Vps39 might trigger a conformational change in Vps41, resulting in a reduced affinity for Apl5. Alternatively, it is possible that HOPS contains additional low-affinity AP-3–binding sites. Using yeast two-hybrid assays, we detected a strong interaction between Apl5 and Vps41 and weak interactions between Apl5 and several of the remaining HOPS subunits (unpublished results). This may explain why binding of Apl5 to Vps11 and Vps18 is attenuated in vps16
Δ cells (B).
Despite the known interactions of Vps39, Vps41, and the vacuole Rab Ypt7 (Wurmser et al., 2000
; Brett et al., 2008
), we found that Ypt7 was not required for AP-3 binding to HOPS in vitro (C). However, differential centrifugation experiments () suggest that Ypt7 is likely to play a role in the docking and fusion of AP-3 vesicles at the vacuole. We also observe that GST-Apl5-ear binds Vam7, a soluble vacuole SNARE, independently of HOPS ( and ). This result was somewhat unexpected as Vam7 has been reported to interact with HOPS (Stroupe et al., 2006
). Although AP-3 binds two other vacuole SNAREs (Vam3 and Nyv1) through their cargo sorting motifs, it is possible that these SNARE–AP-3 interactions also promote docking and fusion at the vacuole.
Rehling et al. (1999)
proposed that AP-3 binds Vps41 to mediate AP-3 vesicle formation at the late Golgi. However, using fluorescence microscopy to examine HOPS and Apl5 localization, we were unable to detect colocalization of HOPS subunits on AP-3 vesicles (B and Supplemental Movies 1 and 2). Similarly, we were unable to detect HOPS subunits on Sec7-positive late Golgi compartments (B and Supplemental Movies 5–7). In contrast, we readily detected foci enriched in Apl5 at late Golgi compartments marked by Sec7 (A and , B and C, and Supplemental Movies 4 and 9). The signal-to-noise ratios in our microscopy experiments are sufficiently high that if Vps41 were to form a clathrin-like outer-shell matrix on AP-3 vesicles, then Vps41 (and perhaps other HOPS subunits) should have been readily detected on AP-3 vesicles, late Golgi compartments, or both.
Also arguing against a strict requirement for Vps41 in AP-3 vesicle formation, we found that a vps41
Δ null mutant accumulates Apl5-GFP puncta that do not colocalize with the late Golgi marker, Sec7 (B), and displays a corresponding decrease in cytosolic Apl5-GFP ( and B). These phenotypes are shared by vam3tsf
cells, which have been reported to accumulate vesicles at nonpermissive temperature (A; Rehling et al., 1999
). The accumulation of Apl5 vesicles is also consistent with differential centrifugation studies of vam3
conditional and null mutants (; Rehling et al., 1999
). These results suggest that both proteins are required for docking of AP-3 vesicles at the vacuole, and this docking event must take place before AP-3 can uncoat from the vesicle and be released back to the cytosol. Although the extent of the Apl5 puncta accumulation in both strains occurs to a similar extent, we noticed decreased mobility of some Apl5-GFP puncta in the vam3tsf
strain at nonpermissive temperatures (Supplemental Movie 8). However, both the accumulation of puncta and the decrease in puncta mobility rapidly disappear upon shifting the cells to permissive temperature (unpublished results). Similar to vam3tsf
mutants, cells expressing the dominant-negative vacuole SNARE Vam7-Qc
5Δ (Schwartz and Merz, 2009
) also accumulate Sec7-negative AP-3 vesicles (our unpublished results). This argues that AP-3 vesicle accumulation is a consequence of defective docking or fusion at the vacuole.
In marked contrast to the docking and fusion mutants, we found that cells lacking the dynamin homolog Vps1 do not accumulate Apl5-GFP puncta (C), suggesting that Apl5-GFP puncta accumulation is not a general consequence of perturbations to AP-3 trafficking. Although our data strongly suggest that Vps41 is not required for AP-3 vesicle budding, we cannot rule out the possibility that AP-3 budding sites at the late Golgi, or post-Golgi AP-3 vesicles, contain substoichiometric amounts of HOPS at levels below the detection threshold, or that Vps41 associates with budding AP-3 vesicles briefly and then very rapidly dissociates. Such a role would be similar to findings that although clathrin is not essential for endocytosis in yeast, it has an assistive role (Baggett and Wendland, 2001
Consistent with vesicle accumulation in strains that contain nonfunctional vacuole fusion machinery, we are able to detect transient Apl5 and HOPS colocalization at the vacuole membrane in wild-type cells (B; Supplemental Movies 1 and 2). Localization of Apl5 puncta at the vacuole is seen in nearly all Apl5-tagged strains, albeit to varying extents depending on strain background. We were also able to see vacuolar Apl5 fluorescence when with the vital stain FM4-64 was used to mark the vacuole (C; Supplemental Movie 3).
Experiments in metazoans imply that physical and functional interactions between HOPS and AP-3 are evolutionarily conserved. Mice with mutations in AP-3 subunits β (pearl
) and δ (mocha
) as well as in the HOPS subunit VPS33A (buff
) display hypopigmentation of the fur as well as blood clotting defects (Kantheti et al., 1998
; Feng et al., 1999
; Suzuki et al., 2003
). Similarly, in Drosophila melanogaster
, mutations in both HOPS and AP-3 result in defective pigment granule biogenesis; the eye pigment mutations deep orange, carnation
, and light
correspond to VPS18, VPS33A, and VPS41, whereas garnet
, and orange
correspond to the AP-3 δ, β, μ, and ς subunits (Ooi et al., 1997
; Warner et al., 1998
; Mullins et al., 1999
; Sevrioukov et al., 1999
; Kretzschmar et al., 2000
; Mullins et al., 2000
). Small interfering RNA knockdown of VPS16A also results in defective pigment granule biogenesis (Pulipparacharuvil et al., 2005
). Furthermore, carnation
, and deep orange
show genetic interactions with the AP-3 δ subunit garnet
(Lloyd et al., 1998
), consistent with a functional interaction between AP-3 and HOPS. Finally, AP-3 and HOPS subunits were shown to coimmunoprecipitate from cultured mammalian cells (Salazar et al., 2008
). This interaction was detected only in the presence of cross-linker, consistent with our finding that AP-3 interactions with HOPS appear to be evanescent. Thus, experimental results from many systems support the idea that AP-3-HOPS interactions are broadly conserved. Because multiple trafficking pathways converge on the vacuole, HOPS may also tether other vesicle adaptors and serve as a general docking nexus at the vacuole, an idea supported by pleiotropic phenotypes in cells lacking HOPS subunits (reviewed in Nickerson et al., 2009
). In this context, it is intriguing that a mammalian HOPS subunit, hVps18, was reported to bind and ubiquitylate GGA3 (Yogosawa et al., 2006
AP-3–HOPS interactions during docking at the vacuole are somewhat unexpected, because coat proteins generally are thought to be shed either during or immediately after budding and as a prerequisite to docking and fusion (reviewed in Bonifacino and Glick, 2004
). However, the COPII inner-shell subunit Sec23 has been shown to bind the TRAPPI (transport protein particle) docking complex at the Golgi (Cai et al., 2007
). Like HOPS, TRAPPI has GEF activity toward its cognate Rab (Jones et al., 2000
; Cai et al., 2007
). Thus, some vesicle adaptors may remain associated with transport vesicles to impart specificity throughout the budding and fusion cycle.