A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae.

A family of glucose transporters mediates glucose uptake in Saccharomyces cerevisiae. We show that the dominant mutation GSF4-1, which impairs glucose repression of SUC2, results in a nonfunctional chimera of the transporters Hxt1p and Hxt4p. Hxt1/4p inhibits the function of wild-type glucose transporters. Similar mutations may facilitate analysis of the major facilitator superfamily.

Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces.

Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction.

Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p.

Mutations in the GSF2 gene cause glucose starvation phenotypes in Saccharomyces cerevisiae. We have isolated the HXT1 gene, which encodes a low-affinity, high-capacity glucose transporter, as a multicopy suppressor of a gsf2 mutation. We show that gsf2 mutants accumulate Hxt1p in the endoplasmic reticulum (ER) and that Gsf2p is a 46-kDa integral membrane protein localized to the ER. gsf2 mutants also display a galactose growth defect and abnormal localization of the galactose transporter Gal2p but are not defective in function or localization of the high-affinity glucose transporter Hxt2p. These findings suggest that Gsf2p functions in the ER to promote the secretion of certain hexose transporters.

Mutations in GSF1 and GSF2 alter glucose signaling in Saccharomyces cerevisiae.

One function of the Saccharomyces cerevisiae Snf1 protein kinase is to relieve glucose repression of SUC, GAL, and other genes in response to glucose depletion. To identify genes that regulate Snf1 kinase activity, we have selected mutants that inappropriately express a SUC2promoter::HIS3 gene fusion when grown in glucose and that require Snf1 function for this phenotype. Mutations representing two new complementation groups (gsf1 and gsf2) were isolated. gsf1 mutations affect two distinct responses to glucose: the Snf1-regulated glucose repression of SUC2 and GAL10 transcription and the Snf1-independent induction by glucose of HXT1 transcription. gsf2 mutations relieve glucose repression of SUC2 and GAL10 transcription and, in combination with snf1 delta, cause an extreme slow growth phenotype. The GSF2 gene was cloned by complementation of the gsf2-1 snf1 delta slow growth phenotype and encodes a previously uncharacterized 46kD protein.

Characterization of HIR1 and HIR2, two genes required for regulation of histone gene transcription in Saccharomyces cerevisiae.

The products of the HIR1 and HIR2 genes have been defined genetically as repressors of histone gene transcription in S. cerevisiae. A mutation in either gene affects cell cycle regulation of three of the four histone gene loci; transcription of these loci occurs throughout the cell cycle and is no longer repressed in response to the inhibition of DNA replication. The same mutations also eliminate autogenous regulation of the HTA1-HTB1 locus by histones H2A and H2B. The HIR1 and HIR2 genes have been isolated, and their roles in the transcriptional regulation of the HTA1-HTB1 locus have been characterized. Neither gene encodes an essential protein, and null alleles derepress HTA1-HTB1 transcription. Both HIR genes are expressed constitutively under conditions that lead to repression or derepression of the HTA1 gene, and neither gene regulates the expression of the other. The sequence of the HIR1 gene predicts an 88-kDa protein with three repeats of a motif found in the G beta subunit of retinal transducin and in a yeast transcriptional repressor, Tup1. The sequence of the HIR2 gene predicts a protein of 98 kDa. Both gene products contain nuclear targeting signals, and the Hir2 protein is localized in the nucleus.

Histone regulatory (hir) mutations suppress delta insertion alleles in Saccharomyces cerevisiae.

Changes in histone gene dosage as well as mutations within some histone genes suppress delta insertion mutations in the HIS4 and LYS2 loci of Saccharomyces cerevisiae by altering the site of transcription initiation. We have found that three histone regulatory (hir) mutations, identified by their effects on the regulation of histone gene expression, suppress the same insertion mutations. In addition, we have examined whether any previously identified spt (suppressor of Ty) mutations might suppress the delta insertion alleles because of effects on histone gene regulation. Our results demonstrate that mutations in the histone genes SPT11/HTA1 and SPT12/HTB1 and in three other SPT genes, SPT1, SPT10 and SPT21, confer Hir- phenotypes. The spt1 mutation was found to be an allele of HIR2 while the spt10 and spt21 mutations are not in any of the known HIR genes.

DNA-binding properties of the Drosophila melanogaster zeste gene product.

The ability of the zeste moiety of beta-galactosidase-zeste fusion proteins synthesized in Escherichia coli to bind specific DNA sequences was examined. Such fusion proteins recognize a region of the white locus upstream of the start of transcription; this region has previously been shown to be required for genetic interaction between the zeste and white loci. Another strong binding site was localized to a region between 50 and 205 nucleotides before the start of the Ubx transcriptional unit; expression of the bithorax complex is also known to be influenced by the zeste locus. Weaker binding sites were also seen in the vicinity of the bxd and Sgs-4 genes, but it is currently unclear whether these binding sites play a role in transvection effects. The DNA-binding activity of the zeste protein is restricted to a domain of approximately 90 amino acids near the N terminus. This domain does not appear to contain homeobox or zinc finger motifs found in other DNA-binding proteins. The DNA-binding domain is not disrupted by any currently characterized zeste mutations.

Nucleotide sequence and structural analysis of the zeste locus of Drosophila melanogaster.

The zeste locus plays a central role in transvection phenomena, where the synaptic pairing of chromosomes carrying genes with which zeste interacts influences the expression of these genes. To explore the possible functions of the zeste gene product in this process, we have determined the DNA sequences both of a fragment of Drosophila genomic DNA capable of rescuing mutant zeste phenotypes, and of a near full-length cDNA clone derived from the 2.4-kb zeste mRNA. These data show that the zeste gene is interrupted by two small introns, and suggest that the majority of zeste sequences are contained within an intron of another transcriptional unit of opposite polarity. A large region of the predicted zeste product is comprised almost exclusively of glutamine and alanine residues. A domain near the N terminus of this protein, which is sufficient for site-specific DNA binding, is highly charged, as is the C-terminal region of the protein. A breakpoint of the rearrangement In (1)e(bx), which is associated with a za-like phenotype, is found within sequences encoding the zeste product, and would produce a truncated protein. The neomorphic mutation zv77h is correlated with a 300-bp deletion of sequences determining the untranslated 5' leader of the zeste messenger, but may also remove the initiating ATG codon, resulting in a zeste protein with an altered N terminus.