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Query: UNIPROT:P06889 (Mol)
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A mutant screen has been designed to isolate mutants in Saccharomyces cerevisiae deficient in spore wall dityrosine. As shown by electron microscopy, most of the mutant spores lacked only the outermost, dityrosine-rich layer of the spore wall. Mutant dit101, however, was additionally lacking the chitosan layer of the spore wall. Chemical measurements showed that this mutant does not synthesize chitosan during sporulation. The mutant spores were viable but sensitive to lytic enzymes (glusulase or zymolyase). Unlike most of the dit-mutants, dit101 did show a distinctive phenotype in vegetative cells: they grew normally but contained very little chitin and were therefore resistant to the toxic chitin-binding dye, Calcofluor White. The cells showed barely detectable staining of the walls with Calcofluor White or primulin. The decrease in the amount of chitin in vegetative cells and the absence of chitosan in spores suggested that the mutant dit101 could be defective in a chitin synthase. Indeed, a genomic yeast clone harboring the gene, CSD2, sharing significant sequence similarity with yeast chitin synthases I and II (C. E. Bulawa (1992), Mol. Cell. Biol. 12, 1764-1776), complemented our mutant and was shown to correspond to the chromosomal locus of dit101. Thus, the mutations dit101 and csd2 (and probably also call; M. H. Valdivieso et al., (1991), J. Cell Biol. 114, 101-109) were shown to be allelic. The gene was mapped to chromosome II and was located about 3 kb distal of GAL1. Using this DNA clone, a transcript of about 3500-4000 nucleotides was detected. Comparing RNA isolated from vegetative cells and from sporulating cells at different times throughout the sporulation process, no significant differences in DIT101 transcript levels could be detected indicating absence of sporulation-specific transcriptional regulation. However, the amount of DIT101 transcript changed significantly at different stages of the mitotic cell cycle, peaking after septum formation, but before cytokinesis. As most of the chitin synthesis of vegetative cells occurs at this stage of the cell division cycle, chitin synthesis mediated by DIT101 could be primarily regulated at the level of transcription in vegetatively growing cells.
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PMID:DIT101 (CSD2, CAL1), a cell cycle-regulated yeast gene required for synthesis of chitin in cell walls and chitosan in spore walls. 129 86

The transcriptional activation function of the Saccharomyces cerevisiae GAL4 protein is modulated by the GAL80 and GAL3 proteins. In the absence of galactose, GAL80 inhibits the function of GAL4, presumably by direct binding to the GAL4 protein. The presence of galactose triggers the relief of the GAL80 block. The key to this relief is the GAL3 protein. How GAL3 and galactose activate GAL4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of GAL3 is the inducer that interacts with GAL80 or the GAL80-GAL4 complex. Here we report that overproduction of the GAL3 protein causes constitutive expression of GAL/MEL genes in the absence of exogenous galactose. Overproduction of the GAL1 protein (galactokinase) also causes constitutivity, consistent with the observations that GAL1 is strikingly similar in amino acid sequence to GAL3 and has GAL3-like induction activity. Cells lacking the GAL10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of GAL3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of GAL1. On the basis of these data, we propose a new model for galactose-induced activation of the GAL4 protein. This model invokes galactose-activation of the GAL3 and GAL1 proteins which in turn elicit an alteration of the GAL80-GAL4 complex to activate GAL4. This model is consistent with all the known features of the system and has important implications for manipulating GAL4-dependent transcriptional activation in vitro.
Mol Cell Biol 1992 Jun
PMID:Overproduction of the GAL1 or GAL3 protein causes galactose-independent activation of the GAL4 protein: evidence for a new model of induction for the yeast GAL/MEL regulon. 131 7

High-level expression of a transpositionally competent Ty1 element fused to the inducible GAL1 promoter on a 2 microns plasmid (pGTy1) overcomes transpositional dormancy in Saccharomyces cerevisiae. To investigate the mechanisms controlling the rate of Ty1 retrotransposition, we quantitated transposition and Ty1 gene products in cells induced and uninduced for expression of pGTy1. The increase in Ty1 transposition was 45- to 125-fold greater than the increase in Ty1 RNA effected by pGTy1 induction. Translational efficiency of Ty1 RNA was not altered in transposition-induced cells, since p190TYA1-TYB1 protein synthesis increased in proportion to steady-state Ty1 RNA levels. Therefore, expression of a pGTy1 element increases the efficiency of Ty1 transposition at a posttranslational level. Galactose induction of pGTy1 enhanced TYA1 protein processing and allowed detection of processed TYB1 proteins, which are normally present at very low levels in uninduced cells. When the ability of genomic Ty1 elements to complement defined mutations in HIS3-marked pGTy1 elements was examined, mutations in the protease domain or certain mutations in the integrase domain failed to be complemented, but mutations in the reverse transcriptase domain were partially complemented by genomic Ty1 elements. Therefore, the activity of Ty1 elements in yeast cells may be limited by the availability of Ty1 protease and possibly integrase. These results suggest that Ty1 transposition is regulated at the level of protein processing and that this regulation is overcome by expression of a pGTy1 element.
Mol Cell Biol 1992 Jun
PMID:Posttranslational control of Ty1 retrotransposition occurs at the level of protein processing. 131 8

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.
Mol Biol Cell 1992 Aug
PMID:Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae. 132 99

A striking feature of the 3'-end regions in polymerase II transcripts of Saccharomyces cerevisiae adjacent to their processing and polyadenylation sites is the lack of well-defined signal elements. Nonetheless, essential signals have seemed to be confined to compact regions in vivo, and we find that a short RNA with only 70 bases of GAL7 sequence upstream and 8 to 10 bases downstream of the poly(A) addition site is processed in vitro, as is an analogous CYC1 pre-RNA. Specific polyadenylation of a precleaved species further delimits the poly(A) signal and rules out obligatory coupling between cleavage and poly(A) addition. Although little proximal and even less distal sequence is required for accurate cleavage with CYC1 and GAL7, we have been unable to identify common features to which processing could be ascribed. We therefore turned to the coregulated set of genes in the galactose cluster (GAL1, GAL7, and GAL10) to assay their corresponding pre-mRNAs in vitro, in hopes of finding a common theme. By contrast to GAL7, short pre-mRNAs corresponding to GAL1 and GAL10 fail to be cleaved detectably, and only much longer transcripts are susceptible to processing. This indicates that signals, even if preserved, are more widely dispersed than the poly(A) addition site, and these results are unchanged whether extracts are from cells grown on glucose or galactose. As a further surprise, RNAs corresponding to the antisense orientation of the 3'-end regions of all three GAL genes are also effective substrates for the processing machinery in vitro. Computer analysis reveals the presence of polydisperse dyad symmetries that might account for these observations.
Mol Cell Biol 1992 Oct
PMID:Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae. 140 19

We have cloned and sequenced the SIN4 gene and determined that SIN4 is identical to TSF3, identified as a negative regulator of GAL1 gene transcription (S. Chen, R.W. West, Jr., S.L. Johnson, H. Gans, and J. Ma, submitted for publication). Yeast strains bearing a sin4 delta null mutation have been constructed and are temperature sensitive for growth and display defects in both negative and positive regulation of transcription. Transcription of the CTS1 gene is reduced in sin4 delta mutants, suggesting that Sin4 functions as a positive transcriptional regulator. Additionally, a Sin4-LexA fusion protein activates transcription from test promoters containing LexA binding sites. The sin4 delta mutant also shows phenotypes common to histone and spt mutants, including suppression of delta insertion mutations in the HIS4 and LYS2 promoters, expression of promoters lacking upstream activation sequence elements, and decreased superhelical density of circular DNA molecules. These results suggest that the sin4 delta mutation may alter the structure of chromatin, and these changes in chromatin structure may affect transcriptional regulation.
Mol Cell Biol 1992 Oct
PMID:Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae. 140 39

The Saccharomyces cerevisiae GCR2 gene affects expression of most of the glycolytic genes. We report the nucleotide sequence of GCR2, which can potentially encode a 58,061-Da protein. There is a small cluster of asparagines near the center and a C-terminal region that would be highly charged but overall neutral. Fairly homologous regions were found between Gcr2 and Gcr1 proteins. To test potential interactions, the genetic method of S. Fields and O. Song (Nature [London] 340:245-246, 1989), which uses protein fusions of candidate gene products with, respectively, the N-terminal DNA-binding domain of Gal4 and the C-terminal activation domain II, assessing restoration of Gal4 function, was used. In a delta gal4 delta gal80 strain, double transformation by plasmids containing, respectively, a Gal4 (transcription-activating region)/Gcr1 fusion and a Gal4 (DNA-binding domain)/Gcr2 fusion activated lacZ expression from an integrated GAL1/lacZ fusion, indicating reconstitution of functional Gal4 through the interaction of Gcr1 and Gcr2 proteins. The Gal4 (transcription-activating region)/Gcr1 fusion protein alone complemented the defects of both gcr1 and gcr2 strains. Furthermore, a Rap1/Gcr2 fusion protein partially complemented the defects of gcr1 strains. These results suggest that Gcr2 has transcriptional activation activity and that the GCR1 and GCR2 gene products function together.
Mol Cell Biol 1992 Sep
PMID:Role of GCR2 in transcriptional activation of yeast glycolytic genes. 150 87

Casein kinase II (CKII) is composed of a catalytic (alpha) and a regulatory (beta) subunit which unite to form an alpha 2 beta 2 holoenzyme. Saccharomyces cerevisiae CKII consists of two distinct catalytic (Sc alpha and Sc alpha') and regulatory (Sc beta and Sc beta') subunits. Simultaneous disruption of the CKA1 and CKA2 genes (encoding the alpha and alpha' subunits, respectively) is lethal. Such double disruptions can be rescued by GAL1, 10-induced expression of the Drosophila alpha and beta subunits (Dm alpha+beta) together or by GAL10-induced expression of the Drosophila alpha subunit (Dm alpha) alone (Padmanabha, R., Chen-Wu, J. L.-P., Hanna, D. E., and Glover, C. V. C. (1990) Mol. Cell. Biol. 10, 4089-4099). Here we report quantitation, purification, and characterization of casein kinase II activity from such rescued strains. Casein kinase II activity from a strain rescued by Dm alpha alone purifies as a free, catalytically active alpha subunit monomer, whereas that from a strain rescued by Dm alpha/beta purifies as a mixture of tetrameric holoenzyme and monomeric alpha subunit. Interestingly, neither Sc beta nor Sc beta' is present at detectable levels in the enzyme obtained from either strain, raising the possibility that rescue by Dm alpha alone may be mediated via the free, monomeric catalytic subunit. Overexpression of total casein kinase II activity from 6- to 18-fold is not toxic and indeed has no overt phenotypic consequences. Production of large amounts of free catalytic subunit also appears to be without effect, even though free catalytic subunit is normally undetectable in S. cerevisiae.
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PMID:Purification and characterization of casein kinase II (CKII) from delta cka1 delta cka2 Saccharomyces cerevisiae rescued by Drosophila CKII subunits. The free catalytic subunit of casein kinase II is not toxic in vivo. 152 8

We have previously shown that nucleosome loss, obtained by repressing histone H4 mRNA synthesis, activates otherwise inactive PHO5, GAL1, and CYC1 gene promoters (fused to the bacterial beta-galactosidase [lacZ] reporter gene) to moderate levels of activity (approximately 2 to 15% of fully induced levels). We now report that nucleosome loss activates the expression of two additional promoters that are normally induced by independent mechanisms: CUP1 (induced by heavy-metal toxicity) and HIS3 (induced by amino acid starvation). Surprisingly, the level of CUP1-lacZ and HIS3-lacZ activation by nucleosome loss approximates fully induced levels of transcription. These CUP1 and HIS3 promoter activities are increased similarly from either episomal or genomic constructs. Our results emphasize the universality of the mechanism by which nucleosome loss activates yeast promoters. Moreover, a comparison of absolute levels of activation for different promoters suggests that activation by nucleosome loss results in a relatively constant level of activation, while levels obtained by normal induction vary considerably. These data argue that nucleosome loss may play a uniquely dominant role in the regulation of certain promoters.
Mol Cell Biol 1992 Apr
PMID:Nucleosome loss activates CUP1 and HIS3 promoters to fully induced levels in the yeast Saccharomyces cerevisiae. 154 16

The uncoupling protein (UCP) from mammalian brown adipose tissue is an integral component of the mitochondrial inner membrane where it dissipates the proton electrochemical gradient. UCP is transported into mitochondria from the cytosol but lacks a cleavable targeting peptide. We have expressed the rat UCP in Saccharomyces cerevisiae and shown that this protein, which is not normally found in yeast, is targeted to the mitochondria where it disrupts mitochondrial function, probably by uncoupling oxidative phosphorylation. The observed growth defect is dependent upon the level of expression of UCP. When the unmodified UCP cDNA is expressed in yeast under the control of the GAL10 promoter no defect in growth is observed. We have inserted the UCP coding sequence behind the strong phosphoglycerate kinase promoter under the control of the GAL1-10 upstream activation site and introduced a yeast consensus sequence (ATAATG) at the translation start site. We have found that UCP expressed in S. cerevisiae is targeted to mitochondria and that its expression induces a marked growth defect on non-fermentable carbon sources in a manner dependent on induction with galactose.
Mol Microbiol 1992 Feb
PMID:Functional expression of the rat brown adipose tissue uncoupling protein in Saccharomyces cerevisiae. 155 49


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