Analysis of the recruitment of the class I myosin Myo5p to endocytic sites in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, dynamic complexes of multiple proteins that assemble in cortical patches at the plasma membrane have been identified as sites of endocytosis. During endocytic vesicle formation, proteins associate and dissociate at these membrane subdomains in a highly defined temporal order. The type I myosin Myo5p is an essential component of the endocytic machinery of S. cerevisiae. The protein has been shown to be localized to the cortical patches in a short time interval, preceding vesicle scission from the plasma membrane. In the present work, we analyze the process of Myo5p recruitment to the endocytic patch. To investigate which domains are required for targeting of the myosin, we used GFP-tagged Myo5p mutants and life cell imaging. Starting with these experiments, we demonstrate the existence of an inhibitory interaction between the Myo5p tail homology 1 (TH1) domain and the most C-terminal portion of the myosin, which includes the SH3 domain. Such interaction precludes the SH3-mediated Myo5p association with Verprolin and Myo5p recruitment to the cortical patch. Using different kinds of experiments, we find strong evidence that the interaction between the Myo5p TH1 domain and the C-terminus is released at the plasma membrane by dissociation of calmodulin from the Myo5p neck domain, which lies immediately upstream of the TH1 domain. Further, we find that calmodulin release increases the affinity of the Myo5p neck and TH1 domains for acidic phospholipids. Based on our results, we propose a model for calmodulin-regulated patch recruitment of Myo5p, whereby calmodulin dissociation from the Myo5p neck at the plasma membrane releases an intramolecular interaction between the Myo5p TH1 domain and the C-terminus, allowing the association of the Myo5p SH3 domain with Verprolin and the interaction of the Myo5p neck and TH1 domains with acidic phospholipids.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Universität Heidelberg

Year

2007

Author dc:creator

• Grötsch, Helga

Contributors dc:contributor

• Wieland, Felix

Repository record source_url

http://www.ub.uni-heidelberg.de/archiv/7346

OAI identifier oai:identifier

oai:archiv.ub.uni-heidelberg.de:7346