A NOVEL FLUORESCENCE-ACTIVATED CELL SORTER-BASED SCREEN FOR YEAST ENDOCYTOSIS MUTANTS IDENTIFIES A YEAST HOMOLOG OF MAMMALIAN EPS15

A complete understanding of the molecular mechanisms of endocytosis re quires the discovery and characterization of the protein machinery tha t mediates this aspect of membrane trafficking. A novel genetic screen was used to identify yeast mutants defective in internalization of bu lk lipid. The fluorescent lipophilic styryl dye FM4-64 was used in con junction with FACS(R) to enrich for yeast mutants that exhibit interna lization defects. Detailed characterization of two of these mutants, d im1-1 and dim2-1, revealed defects in the endocytic pathway. Like othe r yeast endocytosis mutants, the temperature-sensitive dim mutants wer e unable to endocytose FM4-64 or radiolabeled alpha-factor as efficien tly as wild-type cells. In addition, double mutants with either dim1-D elta or dim2-1 and the endocytosis mutants end4-1 or act1-1 displayed synthetic growth defects, indicating that the DIM gene products functi on in a common or parallel endocytic pathway. Complementation cloning of the DIM genes revealed identity of DIM1 to SHE4 and DIM2 to PAN1. P an1p shares homology with the mammalian clathrin adaptor-associated pr otein, eps15. Both proteins contain multiple EH (eps15 homology) domai ns, a motif proposed to mediate protein-protein interactions. Phalloid in labeling of filamentous actin revealed profound defects in the acti n cytoskeleton in both dim mutants. EM analysis revealed that the dim mutants accumulate vesicles and tubulo-vesicular structures reminiscen t of mammalian early endosomes. In addition, the accumulation of novel plasma membrane invaginations where endocytosis is likely to occur we re visualized in the mutants by electron microscopy using cationized f erritin as a marker for the endocytic pathway. This new screening stra tegy demonstrates a role for She4p and Pan1p in endocytosis, and provi des a new general method for the identification of additional endocyto sis mutants.