end3 and end4: Two Mutants Defective in Receptor-mediated and Fluid-phase Endocytosis in Saccharomyces cer isiae

a-factor, one of two peptide hormones responsible for synchronized mating between MATa and MATa-cell types in Saccharomyces cerevisiae, binds to its cell surface receptor and is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). After internalization, a-factor is delivered to the vacuole via vesicular intermediates and degraded there consistent with an endocytic mechanism (Singer, B., and H. Riezman. 1990. J. Cell Biol. 110:1911-1922; Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). We have isolated two mutants that are defective in the internalization process. Both mutations confer a recessive, temperature-sensitive growth phenotype upon cells that cosegregates with their endocytosis defect. Lucifer yellow, a marker for fluid-phase endocytosis, shows accumulation characteristics in the mutants that are similar to the uptake characteristics of 35S-a-factor. The endocytic defect in end4 cells appears immediately upon shift to restrictive temperature and is reversible at permissive temperature if new protein synthesis is allowed. Furthermore, the end4 mutation only affects a-factor internalization and not the later delivery of a-factor to the vacuole. Other vesicle-mediated p ro cesses seem to be normal in end3 and end4 mutants. END3 and END4 are the first genes shown to be necessary for the internalization step of receptor-borne and fluid-phase markers in yeast. NDOCVTOSIS plays a key role in cell physiology, foremost of which is its function in the uptake of micronutrients such as iron (Hemmaplardh and Morgan, 1976; Karin and Mintz, 1981) and cholesterol (Anderson et al., 1977). Other possible roles for endocytosis include the regulation of hormone response by clearance of responsive elements from the membrane (downregulation; Beguinot et al., 1984), recapture of desialylated proteins and lysosomal proteins from extracellular fluids (Spiess, 1990; Kornfeld and Mellman, 1989), antigen processing and presentation (Harding and Unanue, 1989), transcellular transport ofimmunoglobulins (Kulm and Krahenbuhl, 1982), and controlled redistribution of plasma membrane proteins between apical and basolateral surfaces of cells (Bartles et al., 1987; Matter et al., 1990). As is evident in the examples listed above, most of the studies to date have involved the use of mammalian cells. However, endocytosis has also been described in the budding yeast, Saccharomyces cerevisiae (Riezman, 1985). Using Lucifer yellow-CH (LY), 1 a marker for fluid-phase internalization in mammalian cells, we provided the first evidence Susan Raths' present address is Department of Biology, University of Mississippi, Oxford, Mississippi. 1. Abbreviations used in this paper: CPY, carboxypeptidase Y; EMS, ethylmethanesulfonate; LY, Lucifer yellow; ts, temperature sensitive. that the endocytic pathway exists in yeast. LY was shown to accumulate in the yeast vacuole with nonsaturable kinetics under conditions showing time, temperature, and energy dependence. Furthermore, several of the secretory mutants, namely those conditionally defective in the later steps of secretion were also found to be defective in the accumulation of LY, suggesting some physical or operational overlap between the pathways of secretion and endocytosis in yeast. Subsequent studies by Chvatchko et al. (1986) showed that (x-factor, one of the two peptide pheromones secreted by haploid cell types of S. cerevisiae for mating preparation and conjugation, is also internalized, however only when specifically bound to Mata cells. Once internalized, a-factor is delivered to the vacuole via a vesicular intermediate where it is rapidly degraded in a vacuolar protease-dependent process (Chvatchko, 1987; Dulic and Riezman, 1989; Singer and Riezman, 1990). Concurrent with these findings, Jenness and Spatrick (1986) showed that cellular uptake of a-factor is accompanied by a loss of (x-factor binding sites from the plasma membrane. After this initial period of ~downregulation S cultures reaccumulate newly synthesized receptor sites at the cell surface. These two studies provided corroborative evidence for receptor-mediated endocytosis in yeast and in conjunction with the LY results opened the problem to genetic and biochemical dissection using this simple eukaryote. In animal cells several different selection techniques have g) The Rockefeller University Press, 0021-9525/93/01/55/11 $2.00 The Journal of Cell Biology, Volume 120, Number 1, January 1993 55-65 55 on Jne 9, 2017 D ow nladed fom Published January 1, 1993