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The role of Arf3p and Lsb5p at the actin - endocytosis interface in yeast

Reference	BB/C510091/1
Principal Investigator / Supervisor	Professor Kathryn Ayscough
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	225,886
Status	Completed
Type	Research Grant
Start date	01/02/2005
End date	31/01/2008
Duration	36 months

Abstract

We have identified a number of proteins that function at the interface of the actin cytoskeleton and the cellular machinery required for endocytosis. Our studies have been conducted in the yeast Saccharomyces cerevisiae in order to facilitate rapid studies of the most fundamental aspects of this process. Previously we have demonstrated that proteins that associate with the endocytic machinery are also able to regulate the dynamics of cortical actin. Our recent studies have led us to hypothesise that cortical actin is recruited to pre-formed endocytic sites. At these sites it is assembled into structures that are involved in facilitating the endocytic process. The exact mechanism of action of actin is not yet known but a number of studies indicate that it may be involved in at least two steps of the process, most likely the scission event itself, and then in driving the subsequent movement of the vesicle into the cell. Studies from a number of labs also suggest that actin may be involved in subsequent events of membrane trafficking within the cell. Our research has led us to identify a number of proteins that are potentially involved in coupling actin to the endocytic machinery. Relevant to this application are the proteins Lsb5p and Arf3p. Lsb5p is related to the Gga family of membrane trafficking molecules known to function at earlier stages of the secretory pathway. It has an N-terminal VHS domain and a central GAT domain but it does not contain the gamma adaptin ear motif. We have shown that this protein resides primarily at the plasma membrane and that it can interact with the endocytic adaptor Sla1p and with the activator of actin dynamics Las17p (the yeast WASP homologue). More recently we have found an interaction between Lsb5p and yeast Arf3 (homologous to mammalian Arf6). We now wish to study the molecular interactions of Arf3 and Lsb5p in more detail with an aim to understanding their mechanism of action in more detail and to gain greater knowledge of theprocess of endocytosis and how it is regulated. Specifically we will undertake a broad range of approaches both in vivo (fluorescence microscopy, two hybrid analysis, immunoprecipitation) coupled with complementary in vitro techniques (protein purification and binding studies) to gain insight into this fundamental cell process.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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