Selective Microautophagy of Proteasomes is Initiated by ESCRT-0 and Is Promoted by Proteasome Ubiquitylation

Abstract

The proteasome is central to proteolysis by the ubiquitin-proteasome system under normal growth conditions but is itself degraded through macroautophagy under nutrient stress. A recently described AMPK (AMP-activated protein kinase)-regulated ESCRT (endosomal sorting complex required for transport)-dependent microautophagy pathway also regulates proteasome trafficking and degradation in low glucose conditions in yeast. Aberrant proteasomes are more prone to microautophagy, suggesting the ESCRT system fine-tunes proteasome quality control under low glucose stress. Here we uncover additional features of the selective microautophagy of proteasomes. Genetic or pharmacological induction of aberrant proteasomes is associated with increased mono- or oligo-ubiquitylation of proteasome components, which appear to be recognized by ESCRT-0. AMPK controls this pathway in part by regulating the trafficking of ESCRT-0 to the vacuole surface, which also leads to degradation of the Vps27 subunit of ESCRT-0. The Rsp5 ubiquitin ligase contributes to proteasome subunit ubiquitylation, and multiple ubiquitin-binding elements in Vps27 are involved in their recognition. We propose that ESCRT-0 at the vacuole surface recognizes ubiquitylated proteasomes and initiates their microautophagic elimination during glucose depletion.

Species-specific protein-protein interactions govern the humanization of the 20S proteasome in yeast

“…Yeast whole cell extracts were prepared as described previously (Li & Hochstrasser, 2022). Mid- exponential phase yeast cells were washed twice with ice-cold sterile water and frozen in liquid nitrogen.…”

“…Proteasomes were affinity purified from yeast cells expressing Rpn11-3xFLAG as described previously (Li & Hochstrasser, 2022). Briefly, about 7 ml of cell powder from the same samples as used above for non-denaturing PAGE analysis were thawed on ice, resuspended in 10 ml buffer A (50 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM MgCl 2 , 5 mM ATP, Roche complete EDTA-free protease inhibitor: catalog # 11873580001, lot # 53418500), and incubated for 15 min on ice.…”