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Molecular dissection of the internalization step of endocytosis in "Saccharomyces cerevisiae" : Rvs167p and binding partners

Lombardi, Ruben. Molecular dissection of the internalization step of endocytosis in "Saccharomyces cerevisiae" : Rvs167p and binding partners. 2001, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_6100

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Abstract

Several screens performed in Saccharomyces cerevisiae have led to the isolation of a large number of endocytic mutants. Interestingly, the majority of these mutants were shown to be defective in the internalization step. In this PhD thesis we have started to dissect the internalization step of endocytosis on a molecular level by looking for protein-protein interactions among the proteins required for this step. We found that Rvs167p, one of the two yeast amphiphysin homologs, is a central player in this interaction network and therefore focused the following studies on Rvs167p and its binding partners. In a first series of experiments, we demonstrated that Rvs167p and Rvs161p, the two yeast amphiphysin homologs, function together in vivo. The interaction of these proteins is relevant and required for their stability. We next found that Rvs167p interacts with Sla2p/End4p and Myo5p, two proteins involved in the internalization step, and that these three proteins are part of an approximately 600 kDa protein complex most likely involved in regulating the actin cytoskeleton. In a next series of experiments, we showed that Myo5p, a type I myosin, has a second ATP-independent actin binding site via the SH3-domain in its tail. We show that the interaction with actin requires Vrp1p/End5p and is physiologically relevant. Furthermore, we have identified the two protein kinases Pkh1p and Pkh2p, the yeast PDK1 homologs, to be part of a sphingoid base-mediated signaling pathway required for the internalization step of endocytosis. We found that Rvs167p is a substrate for Pkh2p in vitro and show that mutating the phosphorylation site leads to an endocytic defect in vivo suggesting that Rvs167p is one of the downstram effectors of this signaling cascade.
Advisors:Riezman, Howard
Committee Members:Hall, Michael N.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Growth & Development > Biochemistry (Hall)
UniBasel Contributors:Hall, Michael N.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:6100
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:115
Language:English
Identification Number:
Last Modified:22 Jan 2018 15:50
Deposited On:13 Feb 2009 14:36

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