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H Riezman

Publications and source records attributed to H Riezman.

At least 73 records · Page 4Linked to original sources
PubMed1994

Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast.

Inhibition of ceramide synthesis by a fungal metabolite, myriocin, leads to a rapid and specific reduction in the rate of transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi apparatus without affecting transport of soluble or transmembrane proteins. Inhibition of ceramide biosynthesis also quickly blocks remodelling of GPI anchors to their ceramide-containing, mild base-resistant forms. These results suggest that the pool of ceramide is rapidly depleted from early points of the secretory pathway and that its presence at these locations enhances transport of GPI-anchored proteins specifically. A mutant that is resistant to myriocin reverses its effect on GPI-anchored protein transport without reversing its effects on ceramide synthesis and remodelling. Two hypotheses are proposed to explain the role of ceramide in the transport of GPI-anchored proteins.

Acyltransferases↗
PubMed1994

Rapid protein extraction from Saccharomyces cerevisiae.

We have developed and evaluated an easy and rapid method for extraction of proteins from yeast cells for sodium dodecyl sulphate (SDS)-gel electrophoresis and Western blotting. The procedure comprises a centrifugation step to harvest the cells, addition of a sample buffer and heating, then another centrifugation step before applying the extracted proteins found in the supernatant to an SDS gel. It is applicable to the study of large numbers of samples in 1 day. This procedure is easier, quicker, and as efficient as procedures using base and 2-mercaptoethanol, but somewhat less efficient than lysis with glass beads under certain conditions.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes.

Yeast mutants that are defective in acidification of the lysosome-like vacuole are able to grow at pH 5.5, but not at pH 7. Here, we present evidence that endocytosis is required for this low pH-dependent growth and use this observation to develop a screen for mutants defective in endocytosis. By isolating mutants that cannot grow when they lack the 60-kD vacuolar ATPase subunit (encoded by the VAT2 gene), we isolated a number of vat2-synthetic lethal (Vsl-) mutant strains. Seven of the Vsl- mutants are defective in endocytosis. Four of these mutant strains (end8-1, end9-1, end10-1, and end11-1) show altered uptake of the endocytosed ligand, alpha-factor, and three (end12-1, end12-2, and end13-1) are probably defective in transfer of internalized material to the vacuole. Most of the mutations also confer a strong Ts- growth defect. The mutants defective in uptake of alpha-factor sort newly synthesized vacuolar proteins correctly, while those which may be defective in subsequent transport steps secrete at least a fraction of the newly synthesized soluble vacuolar proteins. The mutations that result in a defect in alpha-factor uptake are not allelic to any of the genes previously shown to encode endocytic functions.

Carboxypeptidases↗
PubMed1994

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. The END3 gene was cloned by complementation of the temperature-sensitive growth defect caused by the end3 mutation and the END3 nucleotide sequence was determined. The END3 gene product is a 40-kDa protein that has a putative EF-hand Ca(2+)-binding site, a consensus sequence for the binding of phosphotidylinositol 4,5-bisphosphate (PIP2), and a C-terminal domain containing two homologous regions of 17-19 aa. The EF-hand consensus and the putative PIP2-binding sites are seemingly not required for End3 protein function. In contrast, different portions of the End3p N-terminal domain, and at least one of the two repeated regions in its C-terminus, are required for End3p activity. Disruption of the END3 gene yielded cells with the same phenotype as the original end3 mutant. An end3ts allele was obtained and this allowed us to demonstrate that End3p is specifically involved in the internalization step of endocytosis. In addition, End3p was shown to be required for proper organization of the actin cytoskeleton and for the correct distribution of chitin at the cell surface.

Actin Cytoskeleton↗
PubMed1994

Intracellular transport of GPI-anchored proteins by yeast.

We have investigated the effects of an inhibitor of ceramide biosynthesis on the glycosylphosphatidylinositol (GPI)-anchoring and intracellular transport of the yeast Gas1 protein. No effect on anchor attachment was demonstrable, but a selective delay in transport from the endoplasmic reticulum to the Golgi complex was observed. The compound also blocked remodeling of GPI-anchors from their base-sensitive to base-resistant forms. A recessive mutation was found that caused resistance to the drug, restored transport of Gas1p, but did not restore ceramide biosynthesis in the presence of the inhibitor. Our results suggest that intracellular transport of GPI-anchored proteins is stimulated by new ceramide synthesis. The role of ceramide may be direct or may be through its use in the remodeling of GPI-anchored proteins other than Gas1p. The need for ceramide can be overcome in the mutant strain.

Ceramides↗
PubMed1994

Expression of Mycobacterium leprae 18-kDa antigen in yeast in a GPI-anchored form.

The 18-kDa protein from Mycobacterium leprae is a major target for the immune response in leprosy. We have developed a system to express this antigen in yeast as a fusion protein with the C-terminal region of the yeast membrane protein GAS1, which would render the recombinant protein anchored to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Cells lacking the GAS1 gene and transformed with the hybrid 18-kDa-GAS1 construct express a polypeptide that reacts with an 18-kDa-specific monoclonal antibody. In addition, these cells react with an alpha-CRD antibody after GPI-PLC treatment. The non-transformed cells are negative. These data indicate that our system may be suitable for the expression of foreign proteins in yeast in a GPI-anchored form.

Bacterial Proteins↗
PubMed1993

Partial purification and characterization of early and late endosomes from yeast. Identification of four novel proteins.

Previously (Singer, B., and Riezman, H. (1990) J. Cell Biol. 110, 1911-1922), we provided evidence for the existence of an endocytic intermediate(s) from the yeast Saccharomyces cerevisiae that is responsible for the transport of the pheromone alpha-factor from the plasma membrane 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 isolation procedure to purify early and late endosomes, consisting of differential centrifugation, flotation on a Nycodenz density gradient, and sedimentation density gradient centrifugation on sucrose/D2O. Using internalized 35S-alpha-factor as a marker, the endosomal fractions were substantially enriched over other membranes, except for Golgi elements 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 endosome fractions. By two-dimensional gel analysis we identified more than 20 proteins spots that are highly enriched in the early/late endosomal fractions. N-terminal protein sequencing resulted in the identification of four novel proteins.

Amino Acid Sequence↗
PubMed1993

Analysis of the sequence requirements for glycosylphosphatidylinositol anchoring of Saccharomyces cerevisiae Gas1 protein.

The Saccharomyces cerevisiae Gas1 protein is synthesized as a precursor with a hydrophobic extension at the carboxyl terminus which is removed and replaced with an inositol containing glycolipid that anchors the protein to the plasma membrane. We performed saturation mutagenesis on the anchor attachment site (Asn506) and showed that only a subset of amino acids with small side chains could act as substrates for peptide cleavage and glycolipid addition. After Asn, which is the most efficient anchor attachment site, Ser, Gly, Ala, Asp, and Cys function with decreasing effectiveness. Mutational analysis also revealed that the 2 adjacent amino acids to the carboxyl side of the anchor attachment site are important for efficient anchoring. These two amino acids should have relatively short side chains with the second position being more critical. Analysis of the region between the anchor attachment site and the carboxyl-terminal hydrophobic region indicated that this region may not simply perform a spacer function.

Alleles↗
PubMed1993

Actin and fimbrin are required for the internalization step of endocytosis in yeast.

In Saccharomyces cerevisiae, alpha-factor is internalized by receptor-mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta-tubulin and actin mutant strains, we showed that actin plays a direct role in receptor-mediated internalization of alpha-factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta-tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin-binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha-factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.

Actins↗
PubMed1993

Yeast endocytosis.

The presence of an endocytic pathway in cells from a wide range of species and the conservation of the proteins involved in this process throughout evolution suggest that endocytosis is of fundamental importance for the eukaryotic cell. However, some surprising recent results have shown that both Dictyostelium discoideum and Saccharomyces cerevisiae can live under laboratory conditions with substantially reduced levels of endocytosis. In this review, I concentrate on endocytosis in S. cerevisiae. Recent progress in the study of intermediates of the endocytic pathway and of mutants affecting the endocytic pathway make this organism an interesting model with which to study the mechanism and functions of endocytosis.

Journal Article↗
PubMed1993

Three clathrin-dependent budding steps and cell polarity.

Clathrin plays an important role in the vesicular transport of proteins in all eukaryotes, but the precise steps in which it is involved may not be identical in all of them. Here, I put forward the hypothesis that three distinct clathrin-dependent budding events are common to all eukaryotes and have the following distinctive features: the first requires actin, the second is used for targeting soluble hydrolases from the Golgi to the hydrolytic compartment, and the third uses a tyrosine localization signal to concentrate membrane proteins. I suggest that the latter budding step is found on endosomes in yeast and is used for retrieval of membrane proteins back to the Golgi. Several testable predictions arise from this hypothesis as well as a possible evolutionary scenario concerning the origin of basolateral and apical plasma membranes in multicellular organisms.

Journal Article↗
PubMed1993

end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae.

alpha-factor, one of two peptide hormones responsible for synchronized mating between MATa and MAT alpha-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, alpha-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-alpha-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 alpha-factor internalization and not the later delivery of alpha-factor to the vacuole. Other vesicle-mediated processes 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.

Carboxypeptidases↗
PubMed1993

Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast.

The Saccharomyces cerevisiae alpha-pheromone receptor, a polytopic, G protein-coupled, membrane protein, is internalized after binding of alpha-factor. Mutational analysis suggested that the first 39 residues of the receptor's cytoplasmic tail carries sufficient information for internalization. A point mutation in one of these 39 residues, K337 to R337, renders the receptor nonfunctional for endocytosis. Other residues, D335 and S338, contribute to the efficiency of internalization. When the sequence DAKSS is added onto a severely truncated receptor, endocytosis of the receptor is restored, showing that this sequence functions to mediate or to signal interaction with the endocytic machinery. Analysis of pheromone response and recovery in strains expressing mutant receptors suggests that receptor internalization is not important for response but contributes to recovery from pheromone.

Amino Acid Sequence↗
PubMed1993

Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast.

The YPT7 gene encodes the Saccharomyces cerevisiae homolog of mammalian rab7 protein. Data obtained from studies on a delta ypt7 mutant suggested that Ypt7p is involved in the endocytic pathway in yeast (Wichmann et al., Cell 71, 1131-1142, 1992). We report here that endocytosed pheromone alpha-factor accumulates in late endosomes in delta ypt7 cells, indicating that Ypt7p is involved in the regulation of transport steps from late endosomes to the vacuole. We also show that alpha-factor can be degraded in a PEP4-dependent manner in a prevacuolar/endosomal compartment in delta ypt7 cells, providing independent evidence that the pathways of vacuole biogenesis and endocytosis in yeast may intersect in the endosomal membrane system.

Biological Transport↗
PubMed1992

Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction.

When alpha factor binds to the yeast alpha factor receptor a signal is transmitted via a tripartite G protein that prepares the cell for conjugation. As a result of alpha factor binding the receptor also undergoes a regulated internalization and hyperphosphorylation. Using cells that lack activity of this tripartite G protein, we show that G protein-mediated pheromone signal transduction is not necessary for regulation of receptor internalization or hyperphosphorylation. Therefore, the processes of signal transduction and down regulation can be uncoupled. We propose that binding of alpha factor to its receptor results in a receptor conformation change that permits receptor hyperphosphorylation and interaction with the endocytic machinery.

Down-Regulation↗
PubMed1991

Determinants for glycophospholipid anchoring of the Saccharomyces cerevisiae GAS1 protein to the plasma membrane.

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS1) encoding the 125-kDa protein (Gas1p) and found that the function of Gas1p is not essential for cell viability. The nucleotide sequence of GAS1 predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N- and O-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gas1p is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gas1p depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.

Amino Acid Sequence↗
PubMed1991

Quantitation of alpha-factor internalization and response during the Saccharomyces cerevisiae cell cycle.

The alpha-factor pheromone binds to specific cell surface receptors on Saccharomyces cerevisiae a cells. The pheromone is then internalized, and cell surface receptors are down-regulated. At the same time, a signal is transmitted that causes changes in gene expression and cell cycle arrest. We show that the ability of cells to internalize alpha-factor is constant throughout the cell cycle, a cells are also able to respond to pheromone throughout the cycle even though there is cell cycle modulation of the expression of two pheromone-inducible genes, FUS1 and STE2. Both of these genes are expressed less efficiently near or just after the START point of the cell cycle in response to alpha-factor. For STE2, the basal level of expression is modulated in the same manner.

Cell Cycle↗