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
In animals, endocytosis of a seven-transmembrane GPCR is mediated by arrestins to propagate or arrest cytoplasmic G protein–mediated signaling, depending on the bias of the receptor or ligand, which determines how much one transduction pathway is used compared to another. In
- Award ID(s):
- 1713880
- NSF-PAR ID:
- 10286080
- Publisher / Repository:
- American Association for the Advancement of Science (AAAS)
- Date Published:
- Journal Name:
- Science Signaling
- Volume:
- 14
- Issue:
- 695
- ISSN:
- 1945-0877
- Page Range / eLocation ID:
- Article No. eabe4090
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract 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 ofVPS4 from anend3 Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTHvps4 Δ 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,vps4 Δrcy1 Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM. -
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