Abstract
The lysosome plays an important role in maintaining cellular nutrient homeostasis. Regulation of nutrient storage can occur by the ubiquitination of certain transporters that are then sorted into the lysosome lumen for degradation. To better understand the underlying mechanism of this process, we performed genetic screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, Rapamycin-Induced Degradation (RapiDeg), to test the sorting defects caused by these mutants. These tests revealed that ubiquitinated vacuole membrane proteins recruit ESCRTs to the vacuole surface, where they mediate cargo sorting and direct cargo delivery into the vacuole lumen. Our findings demonstrate that the ESCRTs can function at both the late endosome and the vacuole membrane to mediate cargo sorting and intra-luminal vesicle formation.
Highlights
The lysosome is best known as a degradative organelle, but it plays an important role in maintaining ion and nutrient homeostasis in the cell
Genetic selection for mutants defective in vacuole membrane protein degradation pathway We have previously shown that Ypq1 sorting from the vacuole membrane into the vacuole lumen after lysine withdrawal is ubiquitin dependent
To better understand the vacuole membrane protein sorting pathway, we developed genetic approaches to identify new machinery required for this process
Summary
The lysosome is best known as a degradative organelle, but it plays an important role in maintaining ion and nutrient homeostasis in the cell. The precise regulation of lysosomal membrane proteins is crucial to maintain nutrient and ion balance in the cell. We found that the vacuole membrane lysine transporter Ypq is ubiquitinated after lysine withdrawal by Rsp, a Nedd family HECT-type E3 ubiquitin ligase. Rsp is recruited to the vacuole membrane by Ssh, a PY-motif containing type-I transmembrane protein. Ypq is sorted into the vacuole lumen for degradation (Li et al, 2015b). We reported that the zinc transporter Cot is ubiquitinated by Tul, a RING-type E3 ligase, and is sorted into the vacuole lumen for degradation upon the
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