Cargo induced recruitment of the endocytic adaptor Sla1 and the role of Sla1-clathrin binding in endocytosis
Tolsma, Thomas O., author
Di Pietro, Santiago, advisor
Ross, Eric, committee member
DeLuca, Jennifer, committee member
Reist, Noreen, committee member
Clathrin-mediated endocytosis is a highly dynamic process that is essential in all eukaryotes. This process is utilized for a number of functions including the uptake of extracellular nutrients, manipulation of the plasma membrane content, downregulation of cell signaling pathways, and viral entry. While differences in protein composition, sequence, and structure do exist between species for this process, many core protein functions and the mechanistic steps involved in endocytic vesicle formation and internalization are highly conserved. This has allowed findings from one species to be applicable to another. For this reason Saccharomyces cerevisiae has been characterized as a highly useful model organism for studying and identifying key proteins and conserved mechanisms in clathrin-mediated endocytosis that are found in all eukaryotes. In yeast, roughly 60 proteins have been identified as being part of the endocytic machinery. Clathrin-mediated endocytosis begins with the recruitment of early endocytic proteins that establish the site of endocytosis. This includes scaffolding and coat proteins, such as clathrin, that aggregate at the plasma membrane through interactions with lipids, protein cargo, and other components of the endocytic machinery. This is followed by recruitment of other late coat proteins that further prepare the site for internalization. Following coat formation the mobile phase of membrane invagination is initiated by the recruitment of the actin polymerization machinery. Actin polymerization then generates an inward force at the site of endocytosis that causes invagination of the plasma membrane. The invagination is then separated from the plasma membrane through the recruitment of scission proteins that pinch off the endocytic vesicle. Lastly the internalized vesicle undergoes a process of coat protein disassembly before being targeted to its proper destination in the cell. While much of this process has been well characterized, significant gaps in our understanding of how different steps in endocytic progression are coordinated and how endocytic proteins function still exist. Using a combination of yeast genetics, fluorescent microscopy, electron microscopy, and biochemistry we have furthered our understanding of clathrin-mediated endocytosis, focusing on the role adaptor-clathrin and adaptor-cargo binding plays in formation and progression of the endocytic process. Our work began by focusing on the role of the adaptor protein Sla1, a clathrin and cargo binding protein that serves essential roles in endocytosis. It was previously established that Sla1 binds clathrin through a variable clathrin box of sequence LLDLQ. Loss of clathrin binding by mutation of this clathrin box has a dramatic effect on endocytosis such as an increased patch lifetime of Sla1 at endocytic sites, and dramatic defects in internalization of endocytic protein cargo. While these experiments demonstrated the importance of Sla1-clathrin binding in endocytosis, they did not explain why Sla1-clathrin binding was important and how this interaction contributes mechanistically to endocytic progression. By imaging Sla1 and clathrin, our work demonstrates that Sla1 contributes to proper clathrin recruitment to endocytic sites. A loss of proper recruitment of clathrin to endocytic sites by mutation of the Sla1 variable clathrin box also resulted in significant accumulation of other endocytic proteins, including those involved in actin polymerization. The lifetime of these additional endocytic components lasted for significantly longer at endocytic sites, some having a disruption in normal recruitment dynamics. Despite this accumulation of the actin polymerization machinery, there is a significant delay in actin polymerization and an increase in actin polymerization time and levels at endocytic sites. Our results also demonstrate defects in the formation of the endocytic invagination and delays in scission. Thus, the Sla1-clathrin interaction is needed for normal progression through different stages of the endocytic process. A second question in the endocytic field that has received little attention is the role cargo plays in the recruitment of the endocytic machinery. The conventional view is that first the endocytic machinery forms an endocytic site and then cargo is concentrated by binding adaptor proteins. Sla1 has previously been shown to bind to endocytic protein cargo that contains the amino acid sequence NPFxD through its SHD1 domain. It has also been shown through biochemical experiments that Sla1 binds Ubiquitin via its third SH3 domain. Both NPFxD and Ubiquitin have been shown to be important signals of cargo for entry into the endocytic pathway. The question, however, remained as to whether cargo binding via these signals contributes to recruitment of the adaptor Sla1 to endocytic sites. The work described in this dissertation will present evidence that this is indeed the case.
Includes bibliographical references.
Includes bibliographical references.