AP‐3 vesicle uncoating occurs after HOPS‐dependent vacuole tethering

Abstract

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

“…In unstressed cells, CMAC was uniformly restricted to vacuoles (Figure 5H). However, vacuoles of both wild type and Δaps3 likely underwent homotypic fusion during heat-ramp as cells tended to have a single large vacuole, consistent with previous reports of protective vacuole stress responses after heat (Schoppe et al, 2020). In support of our model, fewer wild type cells retained vacuolar CMAC compared to Δaps3 at 30 min post heat-ramp (Figure 5H).…”

“…Sna2), it is challenging to distinguish which of these sites is the nexus for cell death. However, an enforced-retargeting strategy recently confirmed that Yck3 is dispensable for vesicle formation at origin membranes, and instead functions at the vacuole membrane where Yck3 promotes tethering and SNARE-mediated fusion of vesicles with vacuole membranes (Cabrera et al, 2010;Schoppe et al, 2020). These findings support our model that Yck3 kinase promotes cell death by acting at the vacuole membrane.…”

“…This further implicates its vesicle trafficking function in the dying process following stress. To test this more directly, we investigated the requirement for Arf1, a small GTPase known to be required by human/yeast AP-1 and AP-3 complexes (inferred for yeast AP-3) for docking onto donor membranes to collect cargo (Anand et al, 2009;Nie et al, 2003;Ooi et al, 1998;Schoppe et al, 2020;Seaman et al, 1996). To address the role of yeast Arf1 without disrupting other membrane trafficking pathways requiring Arf1, we designed point mutations in AP-3 to prevent binding to Arf1.…”

“…Similar to mammals, yeast AP-1 and AP-3 adaptor complexes engage their respective cargo proteins by recognizing amino acid sequence motifs present in nascent, recycled or internalized proteins protruding from late/post-Golgi or endosome membranes. After engaging their cargo, AP-1 and AP-3 plus other proteins generate vesicles that transport and deliver their cargo by fusing to a target membrane with the aid of additional factors (Casler and Glick, 2020;Cowles et al, 1997a;Hirst et al, 2001;Schoppe et al, 2020;Vowels and Payne, 1998). Yeast AP-3 traffics from late/post-Golgi membranes directly to the vacuole/lysosome membrane (Cowles et al, 1997a;Odorizzi et al, 1998;Simpson et al, 1997;Stepp et al, 1997), while AP-1 is more important for Golgi and endosome recycling pathways, with a lesser role in trafficking to the vacuole (Buelto et al, 2020;Casler and Glick, 2020;Daboussi et al, 2012).…”

Vtc5 Is Localized to the Vacuole Membrane by the Conserved AP-3 Complex to Regulate Polyphosphate Synthesis in Budding Yeast

“…Alternatively, Vtc5 could be transported in conjunction with other AP-3 cargoes, such as Vam3, which is also required for wild-type levels of polyP accumulation ( 61 ). Regardless, it is likely that Vtc5 delivery requires interaction of AP-3 coated vesicles with Vps41 of the HOPS complex, which facilitates docking and release of cargo into the vacuole membrane ( 52 , 63 – 65 ). In the absence of AP-3, Vtc5 is mislocalized to the vacuole lumen.…”