Plasma membrane to vacuole traffic induced by glucose starvation requires Gga2‐dependent sorting at the trans‐Golgi network

Abstract

Background Information. In the yeast Saccharomyces cerevisiae, acute glucose starvation induces rapid endocytosis followed by vacuolar degradation of many plasma membrane proteins. This process is essential for cell viability, but the regulatory mechanisms that control it remain poorly understood. Under normal growth conditions, a major regulatory decision for endocytic cargo occurs at the trans-Golgi network (TGN) where proteins can recycle back to the plasma membrane or can be recognized by TGN-localised clathrin adaptors that direct them towards the vacuole. However, glucose starvation reduces recycling and alters the localization and post-translational modification of TGN-localised clathrin adaptors. This raises the possibility that during glucose starvation endocytosed proteins are routed to the vacuole by a novel mechanism that bypasses the TGN or does not require TGN-localised clathrin adaptors. Results. Here, we investigate the role of TGN-localised clathrin adaptors in the traffic of several amino acid permeases, including Can1, during glucose starvation. We find that Can1 transits through the TGN after endocytosis in both starved and normal conditions. Can1 and other amino acid permeases require TGN-localised clathrin adaptors for maximal delivery to the vacuole. Furthermore, these permeases are actively sorted to the vacuole, because ectopically forced de-ubiquitination at the TGN results in the recycling of the Tat1 permase in starved cells. Finally, we report that the Mup1 permease requires the clathrin adaptor Gga2 for vacuolar delivery. In contrast, the clathrin adaptor protein complex AP-1 plays a minor role, potentially in retaining permeases in the TGN, but it is otherwise dispensable for vacuolar delivery. Conclusions and significance. This work elucidates one membrane trafficking pathway needed for yeast to respond to acute glucose starvation. It also reveals the functions of TGNlocalised clathrin adaptors in this process. Our results indicate that the same machinery is needed for vacuolar protein sorting at the GN in glucose starved cells as is needed in the presence of glucose. In addition, our findings provide further support for the model that the TGN is a transit point for many endocytosed proteins, and that Gga2 and AP-1 function in distinct pathways at the TGN.

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

“…Gene ontology function analyses of the 84 hits readily identified all four deletion strains for the small (Δaps3), medium (Δapm3), and two large subunits (Δapl5, Δapl6) of the heterotetrameric AP-3 adaptor complex involved in vesicle trafficking to the vacuole/lysosome membrane (Figure 2B). Our screen did not identify knockouts for AP-1 or AP-2 adaptor complexes, or the Gga1 and Gga2 adaptors that are also involved in vacuole trafficking via a separate pathway distinct from AP-3 (Buelto et al, 2020;Casler and Glick, 2020;Daboussi et al, 2012), suggesting an AP-3-specific phenotype.…”

“…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).…”

The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation

“…The Arg permease Can1 is one of the eukaryotic transporters whose downregulation by endocytosis has been best studied. This process was reported to occur under diverse conditions, including the presence of its substrate Arg [ 17 , 29 , 40 ], addition of cycloheximide [ 20 ], several stress conditions [ 41 , 42 ], and starvation for carbon (glucose) [ 26 , 43 ] or nitrogen [ 32 ]. We have recently dissected at the molecular level the mechanism inducing ubiquitylation and endocytosis of Can1 in response to Arg transport.…”

Endosomal cargo recycling mediated by Gpa1 and Phosphatidylinositol-3-Kinase is inhibited by glucose starvation

“…These experiments were performed in media supplemented with the alternative carbon source raffinose, which might provide a distinct osmotic potential that makes direct comparison with the finding that recycling is reduced during carbon withdrawal. Furthermore, the reduced recycling following glucose starvation (Lang et al, 2014) can be bypassed by enforcing cargo ubiquitination in the endomembrane system upstream of MVB sorting (Buelto et al, 2020). This suggests that both the ubiquitin-mediated degradation pathway (MacDonald and Piper, 2016), and the counteracting recycling pathway induced following cargo deubiquitination from ESCRT-localised endosome compartments (MacDonald et al, 2012a(MacDonald et al, , 2015a are both regulated in response to glucose starvation to collectively promote reduced surface activity and increased vacuolar degradation.…”