The histidine permease gene (HIP1) of Saccharomyces cerevisiae

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.     

Molecular identification of high and low affinity receptors for nicotinic acid

Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.     

Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae

The high affinity iron uptake complex in the yeast plasma membrane (PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two-hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer (FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl-terminal domain of each protein contains a four-residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two-hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the approximately 13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain (TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl-terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease (monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a (minimal) heterodimer model of the Fet3p.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.     

Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae contains three heat-inducible hsp70 genes. We have characterized the promoter region of the hsp70 heat shock gene YG100, that also displays a basal level of expression. Deletion of the distal region of the promoter resulted in an 80% drop in the basal level of expression without affecting expression after heat shock. Progressive-deletion analysis suggested that sequences necessary for heat-inducible expression are more proximal, within 233 base pairs of the initiation region. The promoter region of YG100 contains multiple elements related to the Drosophila melanogaster heat shock element (HSE; CnnGAAnnT TCnnG). Deletion of a proximal promoter region containing one element, HSE2, eliminated most of the heat-inducible expression of YG100. The upstream activation site (UAS) of the yeast cytochrome c gene (CYC1) can be substituted by a single copy of HSE2 plus its adjoining nucleotides (UASHS). This hybrid promoter displayed a substantial level of expression before heat shock, and the level of expression was elevated eightfold by heat shock. YG100 sequences that flank UASHS inhibited basal expression of UASHS in the hybrid promoter but not its heat-inducible expression. This inhibition of basal UASHS activity suggests that negative regulation is involved in modulating expression of this yeast heat shock gene.     

Involvement of histidine permease (Hip1p) in manganese transport in Saccharomyces cerevisiae

In a search for components involved in Mn²⁺ homeostasis in the budding yeast Saccharomyces cerevisiae, we isolated a mutant with modifications in Mn²⁺ transport. The mutation was found to be located in HIP1, a gene known to encode a high-affinity permease for histidine. The mutation, designated hip1–272, caused a frameshift that resulted in a stop codon at position 816 of the 1812-bp ORF. This mutation led to Mn²⁺ resistance, whereas the corresponding null mutation did not. Both hip1–272 cells and the null mutant exhibited low tolerance to divalent cations such as Co²⁺, Ni²⁺, Zn²⁺, and Cu²⁺. The Mn²⁺ phenotype was not influenced by supplementary histidine in either mutant, whereas the sensitivity to other divalent cations was alleviated by the addition of histidine. The cellular Mn²⁺ content of the hip1–272 mutant was lower than that of wild type or null mutant, due to increased rates of Mn²⁺ efflux. We propose that Hip1p is involved in Mn²⁺ transport, carrying out a function related to Mn²⁺ export.     

The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1. Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.