ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Abstract

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 ESCRTdependent trafficking on endocytic recycling and the PM. K E Y W O R D S clathrin-independent endocytosis, clathrin-mediated endocytosis, endosomal sorting complex required for transport, epsin N-terminal homology domain, intralumenal vesicle, multivesicular body, plasma membrane, vacuolar protein sorting

Candida albicans ENT2 Contributes to Efficient Endocytosis, Cell Wall Integrity, Filamentation, and Virulence

“…In foundational studies, it was determined that secretory mutants display defects in endocytosis in the model S. cerevisiae (50). Recently, a clear linkage between clathrinmediated endocytosis and the endosomal sorting complex required for transport (ESCRT)-dependent protein sorting pathway has been identified in S. cerevisiae (51). There it was found that members of the ESCRT complex are required for the viability of cells lacking the epsins Ent1p and Ent2p in which one ENTH domain has been reintroduced (51).…”

“…Recently, a clear linkage between clathrinmediated endocytosis and the endosomal sorting complex required for transport (ESCRT)-dependent protein sorting pathway has been identified in S. cerevisiae (51). There it was found that members of the ESCRT complex are required for the viability of cells lacking the epsins Ent1p and Ent2p in which one ENTH domain has been reintroduced (51). In the prevacuolar secretory pathway, S. cerevisiae VPS1 contributes to endocytosis as well (52).…”

Compromised osteogenic effect of exosomes internalized by senescent bone marrow stem cells via endocytoses involving clathrin, macropinocytosis and caveolae

“…As non-senescent cellular environment of BMSCs internalized exosomes exclusively via clathrin-mediated endocytosis, senescent BMSCs required additional involvement of macropinocytosis and caveolae-mediated endocytosis to mediate the internalization of exosomes. Clathrin-mediated endocytosis was reported as the common endocytic channel that play an important role in the positive regulation of many intracellular signaling cascades ( Hoban et al, 2020 ; Johnson et al, 2021 ). Caveolae was the most commonly reported non-clathrin coated plasma membrane buds with flask-shaped invaginations that existed on the surface of many mammalian cell types including smooth muscle, type I pneumocytes, fibroblasts, adipocytes, and endothelial cells ( Nanbo et al, 2013 ).…”

The role of Vps4 in cancer development

“…At present, poly (ADP-ribose) polymerase inhibitors (PARPi) operate through a “synthetic lethality” mechanism with mutant DNA repair pathways genes in cancer cells, and PARPis are widely used in cancer such as ovarian cancer ( 57 ). There are two forms of VPS4 in the human body and recently it is confirmed that VPS4A and VPS4B are essential enzymes for the ESCRT pathway and have no substitution ( 58 ), loss of both is fatal ( 59 ).…”