Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae.

Abstract

Abstract. The role of clathrin in retention of Golgi membrane proteins has been investigated. Prior work showed that a precursor form of the peptide mating pheromone or-factor is secreted by Saccharomyces cerevisiae cells which lack the clathrin heavy chain gene (CHC1). This defect can be accounted for by the observation that the Golgi membrane protein Kex2p, which initiates maturation of o~-factor precursor, is mislocalized to the cell surface of mutant cells. We have examined the localization of two additional Golgi membrane proteins, dipeptidyl aminopeptidase A (DPAP A) and guanosine diphosphatase (GDPase) in clathrin-deficient yeast strains. Our findings indicate that DPAP A is aberrantly transported to the cell surface but GDPase is not. In mutant cells carrying a temperature-sensitive allele of CHC1 (chcl-ts), o~-factor precursor appears in the culture medium within 15 min, and Kex2p and DPAP A reach the cell surface within 30 min, after imposing the nonperrnissive temperature. In contrast to these immediate effects, a growth defect is apparent only after 2 h at the nonpermissive temperature. Also, sorting of the vacuolar membrane protein, alkaline phosphatase, is not affected in chcl-ts cells until 2 h after the temperature shift. A temperature-sensitive mutation which blocks a late stage of the secretory pathway, secl, prevents the appearance of mislocalized Kex2p at the cell surface of chcl-ts cells. We propose that clathrin plays a direct role in the retention of specific proteins in the yeast Golgi apparatus, thereby preventing their transport to the cell surface.

Localization of TGN38 to the trans-Golgi network: involvement of a cytoplasmic tyrosine-containing sequence.

“…By analogy with tyrosine-based internalization signals (Pearse, 1985(Pearse, , 1988Glickman et al, 1989;Beltzer and Spiess, 1991), the TGN localization determinant characterized here may bind to TGN-specific clathrin-associated adaptor molecules, such as 3,-adaptin (Robinson, 1990). Interestingly, a large fraction of the yeast Golgi peptidase kex2p, whose cytoplasmic tail is necessary for Golgi localization, is missorted to the plasma membrane in yeast mutants deficient in clathrin heavy chains (Payne and Schekman, 1989;Seeger and Payne, 1992). Interaction with components of non-clathrin coats of the TGN is also possible.…”

Membrane stress is coupled to a rapid translational control of gene expression in chlorpromazine-treated cells

“…After washing the pellets twice in cold acetone, proteins were solubilized in Laemmli sample buVer plus 5% -mercaptoethanol. Labeling and immunoprecipitation of secreted pro--factor were performed as described (Seeger and Payne 1992). Labeled proteins and labeled immunoprecipitated pro--factor were resolved on SDS-PAGE gels and analyzed by Xuorography.…”

Vps52p, Vps53p, and Vps54p Form a Novel Multisubunit Complex Required for Protein Sorting at the Yeast Late Golgi

“…In vps1 mutants, membrane proteins such as ALP are missorted to the plasma membrane and rely on endocytosis to reach the vacuole (Nothwehr et al, 1995). Clathrin is required for the formation of only one class of vesicles at the TGN, and clathrin temperature-sensitive mutants missort TGN proteins (but not ALP) to the cell surface (Seeger and Payne, 1992).…”

“…In fact, few of the Vps proteins that have been characterized to date are even localized to the TGN. Vps1p is required for the budding of TGN vesicles, as is clathrin (Seeger and Payne, 1992;Nothwehr et al, 1995), but the adaptin subunits that are involved in lysosomal protein sorting in mammalian cells are not absolutely required for CPY sorting; in fact, entry into the CPY pathway does not appear to depend on cytoplasmic tail signals (Roberts et al, 1992;Redding et al, 1996). Therefore, it seems likely that additional proteins exist to control both budding and fusion at the TGN.…”