THE END3 GENE ENCODES A PROTEIN THAT IS REQUIRED FOR THE INTERNALIZATION STEP OF ENDOCYTOSIS AND FOR ACTIN CYTOSKELETON ORGANIZATION IN YEAST

Two Saccharomyces cerevisiae mutants, end3 and end4, defective in the internalization step of endocytosis, have previously been isolated. Th e END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide seq uence was determined. The END3 gene product is a 40-kDa protein that h as a putative EF-hand Ca2+-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-termi nal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not requir ed for End3 protein function. In contrast, different portions of the E nd3p N-terminal domain, and at least one of the two repeated regions i n its C-terminus, are required for End3p activity. Disruption of the E ND3 gene yielded cells with the same phenotype as the original end3 mu tant. An end3(ts) allele was obtained and this allowed us to demonstra te that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper or ganization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.