Abstract

Diverse physiology relies on receptor and transporter protein down-regulation mediated by ESCRTs. Mutations in ESCRT genes are thought to block this process, underlying pathogenesis of human cancers and neurological disorders. However, when orthologous mutations are introduced into model organisms, cells thrive and surface protein down-regulation persists, suggesting other mechanisms compensate for missing ESCRT activity. To better understand this secondary process, we studied degradation of quintessential ESCRT-client proteins (transporter Mup1, receptor Ste3) when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that signaling for endocytosis remains intact, but all proteins aberrantly accumulate on vacuolar lysosome membranes in mutants and some wild type cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, either constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail–safe layer of defense when ESCRTs disregard their clients, representing a two-tiered system that ensures down-regulation of surface polytopic proteins.