Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of ...transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.
In a WT cell (left), endocytosis delivers protein cargoes to late endosomes; given the recently changing views on the structure of the yeast endomembrane system, dotted lines are used here to represent the multiple potential trafficking pathways between multivesicular bodies and the plasma membrane (PM). Cargoes can then be trafficked into vesicles within late endosomes and delivered to the vacuole lumen for degradation. In many endocytosis mutants (center), cargoes accumulate at the PM as a result of decreased endocytic efficiency. In cells that lack Vps4, protein cargoes that are normally trafficked to the vacuole lumen instead accumulate in class E organelles, stacks of membranous tubules adjacent to vacuoles. In the absence of Vps4, internalized cargoes are aberrantly returned to the PM via endocytic recycling. We propose that, when these mutations are combined (right), the redirection of cargo to the PM in an endocytosis mutant, which already has an elevated concentration of cell‐surface proteins, leads to the synthetic lethality observed in this study.
PDF