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.DOI: http://dx.doi.org/10.7554/eLife.26403.001

Retromer is an evolutionarily conserved protein complex, which sorts functionally diverse membrane proteins into recycling tubules/vesicles from the endosome. Many of the identified cargos possess a recycling signal sequence defined as ØX[L/M/V], where Ø is F/Y/W. However, this sequence is present in almost all proteins encoded in the genome. Also, several identified recycling sequences do not follow this rule. How then does retromer precisely select its cargos? Here, we reveal that an additional motif is also required for cargo retrieval. The two distinct motifs form a bipartite recycling signal recognized by the retromer subunits, Vps26 and Vps35. Strikingly, Vps26 utilizes different binding sites depending on the cargo, allowing retromer to recycle different membrane proteins. Thus, retromer interacts with cargos in a more complex manner than previously thought, which facilitates precise cargo recognition.

Receptor‐interacting protein 140 (RIP140) is known as a nuclear co‐repressor for various nuclear receptors. Recently, we identified a PKC epsilon‐triggered post‐translational modification pathway promotes RIP140 cytoplasmic accumulation which in turn blocks GLUT4 trafficking in adipocytes. Here we further examined the roles of cytoplasmic RIP140 in modulating adipokine secretion in adipocytes. We found that silencing RIP140 or PKC epsilon up‐regulated adiponectin secretion but did not affect its mRNA levels. Conditional medium from RIP140‐ or PKC epsilon‐knockdown 3T3‐L1 adipocytes increased glucose uptake in muscle cells as well as decreased glucose production in hepatocytes by promoting AMPK phosphorylation. However, adiponectin neutralized antibody can reverse conditional medium's effect on glucose uptake in muscle cells and glucose production in hepatocytes. Besides, we identified that endothelin‐1 (ET1) enhanced nuclear PKC epsilon activity resulted in RIP140 export from nucleus to cytoplasm, thus exerting its effect on inhibition of adiponectin secretion in adipocytes. Taken together, we demonstred that cytoplasmic RIP140 negatively modulates adiponectin secretion in adipocytes and provided evidence for targeting cytoplasmic RIP140 in adipocytes in promoting systemic insulin sensitivity.NIH grants DK54733, DK60521, DA11190 and K02‐DA13926 to L.‐N.W.

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