PARTIAL-PURIFICATION AND CHARACTERIZATION OF EARLY AND LATE ENDOSOMESFROM YEAST - IDENTIFICATION OF 4 NOVEL PROTEINS

Previously (Singer, B., and Riezman, H. (1990) J. Cell Biol. 110, 1911 -1922), we provided evidence for the existence of an endocytic interme diate(s) from the yeast Saccharomyces cerevisiae that is responsible f or the transport of the pheromone alpha-factor from the plasma membran e to the vacuole. Here we show by kinetic analysis that the endocytic apparatus of yeast is composed of early and late endosomes, similar to what has been found in animal cells. We have developed a three-step i solation procedure to purify early and late endosomes, consisting of d ifferential centrifugation, flotation on a Nycodenz density gradient, and sedimentation density gradient centrifugation on sucrose/D2O. Usin g internalized S-35-alpha-factor as a marker, the endosomal fractions were substantially enriched over other membranes, except for Golgi ele ments and a compartment containing binding protein. These contaminants could not be removed by other standard purification methods. We have analyzed the protein composition of our most pure early and late endos ome fractions. By two-dimensional gel analysis we identified more than 20 protein spots that are highly enriched in the early/late endosomal fractions. N-terminal protein sequencing resulted in the identificati on of four novel proteins.