Abstract
BackgroundThe vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways.ResultsHere we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation.ConclusionsOur data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.
Highlights
The vacuole/lysosome is the final destination of autophagic pathways, but can itself be degraded in whole or in part by selective macroautophagic or microautophagic processes
Degradation of vacuolar membrane proteins in early stationary phase To investigate potential pathways mediating the turnover of the vacuolar membrane, we tagged multiple integral membrane proteins functioning in different processes with green fluorescent protein (GFP), and searched for conditions that could result in their translocation into the vacuolar lumen
It is worth noting that when we used the usual approach of labeling the vacuole with FM4-64 (Fig. 1e), we found that the transport of this lipophilic dye from the plasma membrane (PM) to the vacuolar membrane in early stationary phase was substantially slower than in log phase
Summary
The vacuole/lysosome is the final destination of autophagic pathways, but can itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. For reasons not fully understood, the limiting membrane of vacuole/lysosome, including proteins embedded therein, is not attacked by its own hydrolases under normal conditions, which would otherwise lead to permeablization of the limiting membrane and possibly cell death [6]. Both membrane damage and shift of metabolic activity can trigger the elimination of vacuoles/lysosomes by autophagic pathways, either in whole or in part. In the majority of cases, the autophagy-related (Atg) proteins mediate the generation of autophagosomes from cytoplasmic membrane sources This is true for lysophagy, the elimination of lysosomes by autophagy [7, 8]. Like many other selective autophagy pathways, ubiquitination of lysosomal membrane proteins served as an important signal in the initiation of lysophagy [13,14,15]
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