Mechanisms of Clathrin-Mediated Endocytosis in Budding Yeast

Clathrin-mediated endocytosis (CME) is a major endocytic pathway that is highly conserved among eukaryotes. CME is critical to maintain cell homeostasis, attenuate receptor signaling, generate turnover of proteins at the plasma membrane (PM), and modulate lipid composition of the PM. In the budding yeast S. cerevisiae CME, cargo on the PM is captured, then invaginated into a tubule that pinches into a vesicle to travel into the cell interior. This process relies on the progressive recruitment and coordination of the activities for approximately 60 different proteins. Here, I describe three studies to investigate how the recruitment and activities of endocytic proteins are coordinated during various steps of endoctyosis to ensure successful endocytic events. One way to understand how a complex network of proteins functions is to simplify the process to its most basic components.The Pan1/End3 coat protein complex plays a crucial role in coupling cargo capture to actin assembly, which provides force for PM invagination. I aimed to simplify CME by coupling the upstream coat protein Sla2 to the downstream coat protein Sla1 in order to eliminate the need for the intermediate Pan1/End3 complex. I successfully generated an Sla1-Sla2 fusion protein that was functional for endocytosis. The Sla1-Sla2 fusion protein was recruited to endocytic sites even in the absence of End3 and able to partially rescue some End3 function. However, the Sla1-Sla2 fusion protein was not able to rescue any Pan1 function. These results suggest that Pan1 and End3 have distinct and separable functions in CME. Importantly, generation of this fusion protein is an important initial step in simplifying CME to its minimal functional components.In budding yeast, the N-BAR protein heterodimer, Rvs161/167, plays an important role in scission of the vesicle from the PM. This step is dependent on several additional scission factors. How these factors coordinate with Rvs161/167 to ensure proper scission is poorly understood. Rvs167 contains an N-BAR domain, which contributes to membrane binding and bending, in addition to an SH3 domain. Las17/WASp is a nucleation promoting factor (NPF) that activates Arp2/3-mediated branched actin polymerization, which contributes to vesicle scission. Since Rvs167 and Las17 are predicted to interact through the Rvs167 SH3 domain. I investigated a possible role for Rvs167 in regulation of actin assembly at endocytic sites. I found that Rvs167 can enhance or inhibit Las17 activated Arp2/3-mediated actin assembly in vitro, but found no evidence that this regulation occurs in vivo. To identify a potential role for SH3-mediated protein interactions in vivo, I generated truncation mutants of Rvs167 that lacked the SH3 domain to analyze their recruitment to endocytic sites and function. I found that the N-BAR domain alone is sufficient for Rvs167 recruitment to endocytic sites. However, I found that the N-BAR domain did not rescue negative genetic interactions between rvs167∆ and other endocytic mutations. These results reveal that while not important for localization to endocytic sites, SH3-mediated protein interactions might be important for Rvs167 function in endocytosis. To better understand vesicle scission, it is important to understand how lipid composition changes. I investigated a potential role for the phosphatidate phosphatase (PAP), App1 in regulating endocytosis. PAPs are enzymes that catalyze the conversion of phosphatidic acid (PA) to diacylglycerol (DAG). The relative concentration of these lipids is important for regulation of lipid composition as well as regulation of membrane fission/fusion events. Since App1 has numerous genetic and physical interactions with endocytic factors, I investigated its localization and effects on endocytic dynamics. I found that App1-3GFP signal is predominately cytoplasmic with enrichment on mitochondria. In cells lacking App1, I determined there were subtle defects in endocytic vesicle scission. Overall, these results do not support a role for specific lipid regulation by App1 at endocytic sites.These studies in S. cerevisiae provide insights into the mechanistic nuances of how protein recruitment and activity are coordinated in CME and provide a foundation for future work