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

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.

Bioluminescence imaging of G protein-coupled receptor activation in living mice.

G protein-coupled receptors (GPCRs), a superfamily of cell-surface receptors involved in virtually all physiological processes, are the major target class for approved drugs. Imaging GPCR activation in real time in living animals would provide a powerful way to study their role in biology and disease. Here, we describe a mouse model that enables the bioluminescent detection of GPCR activation in real time by utilizing the clinically important GPCR, sphingosine-1-phosphate receptor 1 (S1P1). A synthetic S1P1 signaling pathway, designed to report the interaction between S1P1 and ß-arrestin2 via the firefly split luciferase fragment complementation system, is genetically encoded in these mice. Upon receptor activation and subsequent ß-arrestin2 recruitment, an active luciferase enzyme complex is produced, which can be detected by in vivo bioluminescence imaging. This imaging strategy reveals the dynamics and spatial specificity of S1P1 activation in normal and pathophysiologic contexts in vivo and can be applied to other GPCRs.