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Rehman, Michael (2011): Analysis of endocytosis at eisosomes. Dissertation, LMU München: Faculty of Biology
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Abstract

The yeast plasma membrane contains at least three microdomains – membrane compartment containing Pma1 (MCP), membrane compartment containing TORC2 (MCT) and membrane compartment containing Can1 (MCC). Eisosomes underlie the MCC domain defined by the marker protein arginine permease (Can1). Eisosomes are large protein assemblies composed of Pil1 and Lsp1 proteins, of which Pil1 is essential for plasma membrane organization. We found that the uncharacterized AAA-ATPase protein Yta6 dynamically colocalizes with eisosomes. Yta6 physically interacts with eisosome components, specifically with Pil1. In PIL1 deletion cells, Yta6 is unable to localize normally to the plasma membrane. Yta6 foci colocalize with the intermediates of FM4-64 on the plasma membrane. The number of these intermediates is increased upon overexpression of Yta6. Overexpressed Yta6 is also able to rescue the defects of endocytosis in cells devoid of amphiphysins. Together rescue experiments and colocalization of a protein cargo Hxt3 with eisosomes suggest that Yta6 likely plays a role in endocytosis at eisosomes. To identify genes whose products function together with eisosome components, we independently carried out a genetic interaction study (epistatic mini array profile) which revealed the protein Emp70. EMP70 showed the strongest correlation in genetic profile with PIL1. Emp70 localizes to a subset of eisosomes in addition to its localization in endosomes and vacuoles. We found eisosomes are required for normal numbers of Emp70 plasma membrane foci. Deletion of Emp70 misdirected endosomal protein Kex2 to vacuole, implicating its essential role in maintaining the architecture of the endosomal compartment. In summary, Yta6 likely plays a role in initiation of endocytosis at eisosomes and Emp70 during intracellular trafficking from plasma membrane to vacuole.