The role of Sla1p in linking actin dynamics to the endocytic machinery in Saccaromyces cerevisiae

Warren, Derek (2002) The role of Sla1p in linking actin dynamics to the endocytic machinery in Saccaromyces cerevisiae. PhD thesis, University of Glasgow.

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Abstract

Slalp is a protein required for cortical actin patch structure and organisation in budding yeast. An association with the endocytic machinery has also been shown by immunoprecipitation of Slalp with EH domain containing proteins, End3p and Panlp. Here we use immunofluorescence to demonstrate that localisation of Slalp to the cell cortex requires its C-terminal repeat region and also End3p. We show that cells either lacking SLAl or expressing the slal?Ct mutant display defects in both fluid phase and receptor mediated endocytosis, suggesting that Slalp may be part of the endocytic machinery. This study also demonstrates modification of Slalp by the Actin Regulating Kinases, Arklp/Prklp and the yeast protein phosphatase-1 Glc7p. Immunofluorescence microscopy revealed that the association of Slalp with cortical actin patches is regulated by the interactions of the Arklp/Prklp kinases. We also provide evidence for the existence of multiple isoforms of Slalp in vivo. Finally, using the yeast 2-hybrid system, we identified Ycl034wp and Ynr065cp as interacting partners of the central region of Slalp. Sequence analysis of Ycl034wp predicted this protein contained a VHS and a GAT domain. These domains have been identified in a number of proteins involved in vesicle trafficking. The mammalian homologues of Ynr065cp (Sortilin and Sorl-1) have also been implicated in vesicle trafficking. This indicates that the central region of Slalp interacts with proteins involved in the transport of vesicles. Taken together, the data presented here allows us to predict a dynamic model where Slalp is able to interact with proteins involved in actin dynamics, the endocytic machinery and also proteins involved in vesicle trafficking. Association of these different sets of machinery may be regulated by cycles of phosphorylation and dephosphorylation. This allows Slalp to function as an adaptor protein, bringing proteins involved in different stages of the endocytic pathway into close proximity and allowing efficient endocytosis.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Kathryn Ayscough
Keywords: Molecular biology, Microbiology
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-72623
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06
URI: http://theses.gla.ac.uk/id/eprint/72623

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