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V L MacKay

Publications and source records attributed to V L MacKay.

At least 19 recordsLinked to original sources
PubMed1995

N- and O-glycosylation and phosphorylation of the bar secretion leader derived from the barrier protease of Saccharomyces cerevisiae.

A secretion leader derived from a domain of the extracellular Barrier protease of the yeast Saccharomyces cerevisiae has been expressed in wild-type and in mnn1, mnn9, and mnn1 mnn9 glycosylation mutant strains of S. cerevisiae. Structural comparison of the extracellular leader by mass spectrometry, peptide mapping, and elementary analysis proved that all strains produced a heterogeneous, heavily glycosylated polypeptide of 161 amino acids with both N- and O-glycosylation and phosphorylation. All three potential Asn N-linked sites were glycosylated to some extent with the expected structures. Neither the different growth media used nor the glycosylation mutations had significant effect on O-glycosylation with respect to both site selectivity and size of the carbohydrate structures. All 33 Ser and 21 Thr residues in the polypeptide were glycosylated at least partially, with an average of more than 2 mannoses/site. Although the mnn1 mutation blocks addition of alpha 1,3-linked mannose, the bar secretion domain expressed in the mnn1 and mnn1 mnn9 transformants unexpectedly contained some O-linked structures with at least 4 mannoses/chain. These O-linked structures were as large as when the leader was expressed in the mnn9 and wild-type strains. The bar secretion domain also had a previously undocumented phosphorylated O-linked structure.

Amino Acid Sequence↗
PubMed1995

A functionally compromised intermediate in extrathymic CD8+ T cell deletion.

We have established a model system for analyzing the induction of self-tolerance among mature peripheral T cells in V beta 5 TCR Tg mice. Both CD4+V beta 5+ and CD8+ V beta 5+ cells undergo a superantigen-driven chronic deletion in the periphery of I-E mice. Prior to their disappearance, CD4+ transgene-expressing cells are activated and then rendered anergic to further stimulation through their TCRs. This scenario differs strikingly in the CD8+ cellular compartment, which is characterized by a distinct population of CD8loV beta 5lo cells localized to the blood and spleen. CD8lo cells are small, express the surface phenotype of memory cells, and rapidly incorporate BrdU in vivo. The kinetics of their appearance and disappearance in adult thymectomized mice, the rapid chasing of BrdU from labeled cells, and their in vivo cortisone sensitivity all suggest CD8lo cells are slated for deletion. Furthermore, their functional incompetence can be documented in vitro in the absence of internucleosomal DNA fragmentation. Thus, we have identified an intermediate population of T cells targeted for peripheral deletion that, although functionally compromised, has not yet undergone programmed cell death.

Animals↗
PubMed1994

Cloning and analysis of the Saccharomyces cerevisiae MNN9 and MNN1 genes required for complex glycosylation of secreted proteins.

Proteins secreted by the yeast Saccharomyces cerevisiae are usually modified by the addition at asparagine-linked glycosylation sites of large heterogeneous mannan units that are highly immunogenic. Secreted proteins from mnn1 mnn9 mutant strains, in contrast, have homogeneous Man10GlcNAc2 oligosaccharides that lack the immunogenic alpha 1,3-mannose linkages. We have cloned and sequenced the MNN9 and MNN1 genes, both of which encode proteins with the characteristics of type II membrane proteins. Mnn9p is a membrane-associated protein with unknown function that is required for the addition of the long alpha 1,6-mannose backbone of the complex mannan, whereas Mnn1p is most likely the alpha 1,3-mannosyltransferase located in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Clathrin-dependent localization of alpha 1,3 mannosyltransferase to the Golgi complex of Saccharomyces cerevisiae.

Posttranslational modification of yeast glycoproteins with alpha 1,3-linked mannose is initiated within a Golgi compartment analogous to the medial Golgi cisternae of higher eukaryotes. We have characterized the synthesis, posttranslational modification, and localization of the yeast alpha 1,3 mannosyltransferase (Mnn1p) using antibodies prepared against a segment of this protein expressed in bacteria. Mnn1p is initially synthesized as a 98.5-kD, type II integral membrane glycoprotein that is modified with both N- and O-linked oligosaccharides. It is subject to a slow, incremental increase in molecular mass that is dependent upon protein transport to the Golgi complex. Self-modification of Mnn1p with alpha 1,3 mannose epitopes, primarily on O-linked oligosaccharides, is at least partly responsible for the incremental increase in molecular mass. Mnn1p is a resident protein of the Golgi complex and colocalizes with guanosine diphosphatase to at least two physically distinct Golgi compartments by sucrose gradient fractionation, one of which may be a late Golgi compartment that also contains the Kex2 endopeptidase. Surprisingly, we found that a significant fraction of Mnn1p is mislocalized to the plasma membrane in a clathrin heavy chain temperature sensitive mutant while guanosine diphosphatase remains intracellular. A mutant Mnn1p that lacks the NH2-terminal cytoplasmic tail is properly localized to the Golgi complex, indicating that clathrin does not mediate Mnnlp Golgi retention by a direct interaction with the Mnn1p cytoplasmic tail. These results indicate that clathrin plays a broader role in the localization of Golgi proteins than anticipated.

Amino Acid Sequence↗
PubMed1989

The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein.

The STE4 and STE18 genes are required for haploid yeast cell mating. Sequencing of the cloned genes revealed that the STE4 polypeptide shows extensive homology to the beta subunits of mammalian G proteins, while the STE18 polypeptide shows weak similarity to the gamma subunit of transducin. Null mutations in either gene can suppress the haploid-specific cell-cycle arrest caused by mutations in the SCG1 gene (previously shown to encode a protein with similarity to the alpha subunit of G proteins). We propose that the products of the STE4 and STE18 genes comprise the beta and gamma subunits of a G protein complex coupled to the mating pheromone receptors. The genetic data suggest pheromone-receptor binding leads to the dissociation of the alpha subunit from beta gamma (as shown for mammalian G proteins), and the free beta gamma element initiates the pheromone response.

Amino Acid Sequence↗
PubMed1988

The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin.

Saccharomyces cerevisiae a cells secrete an extracellular protein, called "barrier" activity, that acts as an antagonist of alpha factor, the peptide mating pheromone produced by mating-type alpha cells. We report here the DNA sequence of BAR1, the structural gene for barrier activity. The deduced primary translation product of 587 amino acids has a putative signal peptide, nine potential asparagine-linked glycosylation sites, and marked sequence similarity of the first two-thirds of the protein with pepsin-like proteases. Barrier activity was abolished by in vitro mutation of an aspartic acid predicted from this sequence homology to be in the active site. Therefore, barrier protein is probably a protease that cleaves alpha factor. The sequence similarity suggests that the first two-thirds of the barrier protein is organized into two distinct structural domains like those of the pepsin-like proteases. However, the BAR1 gene product has a third carboxyl-terminal domain of unknown function; deletion of at least 166 of the 191 amino acids of this region has no significant effect on barrier activity.

Amino Acid Sequence↗
PubMed1987

A yeast operator overlaps an upstream activation site.

The product of the BAR1 gene of Saccharomyces cerevisiae is synthesized only in the a cell type and inactivates alpha-factor, the mating pheromone made by alpha cells. The MAT alpha 2 protein represses the transcription of a-cell-specific genes, including BAR1, in alpha and a/alpha diploid cells. Transcription of BAR1 in a cells in stimulated upon exposure to alpha-factor. Deletion analysis of the 5' noncoding region of the BAR1 gene revealed that the major upstream activation site (UAS) overlaps the 31 bp operator sequence required for MAT alpha 2 repression. This result has implications for the negative control of transcription in yeast. The deletion analysis also indicated that the sequence TGAAACA mediates alpha-factor stimulation.

Base Sequence↗
PubMed1986

Multiple regulation of STE2, a mating-type-specific gene of Saccharomyces cerevisiae.

The Saccharomyces cerevisiae STE2 gene, which is required for pheromone response and conjugation specifically in mating-type a cells, was cloned by complementation of the ste2 mutation. Transcription of STE2 is repressed by the MAT alpha 2 gene product, so that the 1.4-kilobase STE2 RNA is detected only in a or mat alpha 2 strains, not in alpha or a/alpha cells. However, STE2 RNA levels are also increased by the mating pheromone alpha-factor and decreased in strains bearing mutations in the nonspecific STE4 gene. Regulation of STE2 expression in a cells is therefore achieved by several mechanisms.

Base Sequence↗
PubMed1982

Reproducible and rapid methods for the isolation and assay of a-factor, a yeast mating hormone.

The Saccharomyces cerevisiae mating hormone a-factor has been difficult to isolate reproducibly in sufficient yields by methods using ion-exchange chromatography, probably because of its pronounced hydrophobicity. In this work, a hydrophobic adsorbent (Amberlite XAD-2), in an insoluble bead from, was used to isolate larger (up to sixfold greater than previous reports) and quite reproducible (12% standard deviation) quantities of a-factor by adsorption from cell-free filtrates of a cultures. Moreover, when the beads were added to the cultures at the time of inoculation, sixfold greater yields were obtained than when a-factor was adsorbed to the beads from cell-free filtrates. a-Factor was readily eluted from the beads with 1-propanol. The same adsorbent could also be used in the partial purification of the less hydrophobic alpha-factor. Adsorption of both hormones by Amberlite XAD-2 gave a degree of purification comparable to that obtained by the first steps of previously published methods while providing larger yields of hormones. The present procedure is shorter, simpler, and, for a-factor, more reproducible. The activities of both hormones were quantitated by using an assay in which the size distribution of cells in the population was monitored after the addition of hormone of the opposite mating type. The extent of increase in cell size which accompanies hormone treatment is a function of the hormone concentration. To ensure solubilization of a-factor in the aqueous bioassay system, samples were diluted into bovine serum albumin solutions and sonicated before assaying. The resulting assay is most sensitive at hormone concentrations between 0.05 and 2 U/ml, can reliably detect as little as 0.16 ng of hormone, gave results reproducible within 16%, and is convenient for a large number (>100) of samples.

Adsorption↗
PubMed1981

SUC1 gene of Saccharomyces: a structural gene for the large (glycoprotein) and small (carbohydrate-free) forms of invertase.

Saccharomyces cerevisiae revertant strain D10-ER1 has been shown to contain thermosensitive forms of the large (glycoprotein) and small (carbohydrate-free) invertases and a very low level of the small enzyme, along with a wild-type level of the large form (T. Mizunaga et al., Mol. Cell. Biol. 1:460-468, 1981). These characteristics cosegregated in crosses of the revertant strain with wild-type sucrose-fermenting (SUC1) or nonfermenting (suc0) strains. In addition, there is tight linkage between sucrose and maltose fermentation in revertant D10-ER1 (characteristic of the SUC1 and MAL1 genes). From this we infer that a single reversion event is responsible for the several changes observed in D10-ER1, and that this mutation maps within or very close to the SUC1 gene present in the ancestor strain 4059-358D. The revertant SUC1 allele in D10-ER1 (termed SUC1-R1) was expressed independently of the wild-type SUC1 gene when both were present in diploid cells. Diploids carrying only the wild-type or the mutant genes synthesized invertases with the characteristics of the parental Suc+ haploids. The possibility that a modifier gene was responsible for the alterations in the invertases of revertant D10-ER1 was ruled out by appropriate crosses. We conclude that SUC1 is a structural gene that codes for both the large and the small forms of invertase and suggest that SUC2 through SUC5 are structural genes as well.

Alleles↗
PubMed1979

Sexual conjugation in yeast. Cell surface changes in response to the action of mating hormones.

In the yeast Saccharomyces cerevisiae, sexual conjugation between haploid cells of opposite mating type results in the formation of a diploid zygote. When treated with fluorescently labeled concanavalin A, a zygote stains nonuniformly, with the greatest fluorescence occurring at the conjugation bridge between the two haploid parents. In the mating mixture, unconjugated haploid cells often elongate to pear-shaped forms ("shmoos") which likewise exhibit asymmetric staining with the most intense fluorescence at the growing end. Shmoo formation can be induced in cells of one mating type by the addition of a hormone secreted by cells of the opposite mating type; such shmoos also stain asymmetrically. In nearly all cases, the nonmating mutants that were examined stained uniformly after incubation with the appropriate hormone. Asymmetric staining is not observed with vegetative cells, even those that are budded. These results suggest that, before and during conjugation, localized cell surface changes occur in cells of both mating types; the surface alterations facilitate fusion and are apparently mediated by the hormones in a manner that is mating-type specific.

Cell Wall↗
PubMed1977

a-Factor from Saccharomyces cerevisiae: partial characterization of a mating hormone produced by cells of mating type a.

Conjugation between haploid cells of Saccharomyces cerevisiae is mediated through the action of diffusible mating hormones, two of which have been designated as a-factor and alpha-factor. Partially purified fractions exhibiting a-factor activity have been obtained from culture filtrates of a cells by ultrafiltration, ion-exchange chromatography, and gel filtration. The a-factor preparations specifically caused both G1 arrest and morphological alterations in cells of alpha-mating type, whereas a cells, a/alpha diploids, and nonmating alpha mutants were not affected. The a-factor activity was found in the culture filtrates of all a strains tested, but not in filtrates of alpha or a/alpha cell cultures. The hormone is sensitive to various proteases, showing that it is associated with a peptide or protein. Gel filtration studies suggest an apparent molecular weight greater than 600,000; however, this result may be due to aggregation with carbohydrate present in the preparations. Although the biological activities of a-factor are analogous to those described previously for alpha-factor, the chemical properties of these two hormones appear to be quite different.

Cell Division↗
PubMedDate not supplied

Induction of yeast mating pheromone a-factor by alpha cells.

Saccharomyces cerevisiae cells of a and alpha mating types constitutively secrete cell-specific peptide mating pheromones. a-Factor is secreted by a cells and acts on alpha cells, while alpha-factor is secreted by alpha cells and acts on a cells. Confirming preliminary studies, we demonstrate here that cultures of a cells contain higher than constitutive levels of a-factor activity when grown with alpha cells or alpha-factor. This induction of a-factor may result from increased synthesis or increased secretion of a-factor, as opposed to modification or stabilization of preexisting a-factor, as part of the a cell response to alpha-factor, as an a ste2 mutant (which cannot respond to alpha-factor) is not induced by alpha-factor. In mixed cultures inoculated with equal numbers of a cells and alpha cells, a cells predominate by stationary phase. Thus, a series of sequential interactions between a and alpha cells may be involved in establishing optimal hormone concentrations and cell ratios for conjugation.

Animals↗