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.

中文翻译：

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酵母网格蛋白介导的胞吞突变体的生存能力需要依赖ESCRT的蛋白质分选。

胞吞作用调节许多过程，包括信号传导途径，营养吸收和蛋白质更新。在网格蛋白介导的内吞作用（CME）中，衔接子与跨膜货物蛋白的胞质区域结合，许多内吞衔接子也直接参与网格蛋白的募集。该网格蛋白相关的分选蛋白家族包括酵母epsins Ent1 / 2和AP180 / PICALM同源物Yap1801 / 2。缺少这四个衔接子，但表达生存力必需的epsin N末端同源性（ENTH）结构域（4Δ+ ENTH）的突变株表现出内吞缺陷，例如质膜（PM）上的货物堆积。此CME缺陷菌株为揭示与网格蛋白衔接子相互作用的细胞成分提供了理想的背景。我们进行了诱变筛选，以使用菌落扇形报告基因分析鉴定在4Δ+ ENTH细胞中具有致死性的等位基因。通过互补分离候选合成致死基因后，我们证实了VPS4中的突变导致了4Δ+ ENTH菌株的生存。Vps4介导运输（ESCRT）依赖的运输所需的内体分类复合物的最后一步，我们发现多个ESCRT在4Δ+ ENTH细胞中也必不可少，包括Snf7，Snf8和Vps36。从另一个CME突变体end3Δ株中删除VPS4同样导致不可行，并且网格蛋白非依赖性胞吞途径的上调拯救了4Δ+ ENTHvps4Δ细胞。由于野生型背景中Vps4的丢失，导致多个货物在PM处积聚，因为内化蛋白向细胞表面的依赖Rcy1的循环增加。此外，vps4Δrcy1Δ突变体表现出有害的生长表型。在一起，我们的发现揭示了以前不受干扰的ESCRT依赖的贩运对内吞再循环和PM的未意识到的影响。