Transcriptional regulation of YML083c under aerobic and anaerobic conditions.

YML083c and DAN1 were among the Saccharomyces cerevisiae ORFs that displayed the strongest increase in transcript abundance during anaerobic growth compared to aerobic growth, as determined by oligonucleotide microarrays. We here report that transcription of YML083c is regulated by at least three different factors. First, repression under aerobic conditions depends on the presence of heme. Second, deletion analysis of the 5'-flanking region of YML083c and DAN1 revealed two regions responsible for anaerobic induction. Each of these regions conferred anoxia-regulated expression to the heterologous, minimal, CYC1-lacZ reporter. Mutations in the AAACGA subelement, common to the positive acting regions of YML083c and DAN1, almost completely abolished the ability to drive anaerobic expression of the reporter gene. This subelement is similar to the AR1 site, which is involved in anaerobic induction of the DAN/TIR genes. Activation through the AR1 site depends on Upc2. Indeed, transcription from the YML083c promoter was decreased in an upc2 null mutant. Third, expression of Sut1 under aerobic conditions enhanced transcription of YML083c, suggesting that aerobic repression of YML083c is promoted by the general Tup1-Ssn6 co-repressor complex. However, despite the presence of a sequence that matches the consensus for binding of Rox1, YML083c is not controlled by Rox1, since deletion or replacement of the putative binding site did not cause aerobic derepression. Moreover, YML083c expression was undetectable in aerobically grown cells of a rox1 null mutant.

SUT1p interaction with Cyc8p(Ssn6p) relieves hypoxic genes from Cyc8p-Tup1p repression in Saccharomyces cerevisiae.

SUT1 is a hypoxic gene encoding a nuclear protein that belongs to the Zn[II]2Cys-6 family. It has been shown that constitutive expression of SUT1 induces exogenous sterol uptake in aerobically growing Saccharomyces cerevisiae cells. A differential display approach was used to identify genes whose transcription is modified upon SUT1 induction. Within the promoter sequence of one of these genes, DAN1, we identified the region responsive to SUT1 and showed that it has a strong repressive activity when cloned in the vicinity of distinct promoters. Upon SUT1 constitutive expression in aerobiosis, the repression is released, allowing enhanced transcription of the reporter gene. We provide evidence that the repression is promoted by the Cyc8p(Ssn6p)-Tup1p co-repressor and that release of repression is the result of a physical interaction between Sut1p and Cyc8p. Moreover, genetic data suggest that complete derepression of the reporter gene requires a functional Cyc8p. In addition, we show that Sut1p is involved in the induction of hypoxic gene transcription when the cells are shifted from aerobiosis to anaerobiosis.

SUT1 is a putative Zn[II]2Cys6-transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells.

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

Disruption and functional analysis of six ORFs on chromosome XII of saccharomyces cerevisiae: YLR124w, YLR125w, YLR126c, YLR127c, YLR128w and YLR129w.

In the context of the EUROFAN programme, we report the deletion and functional analysis of six open reading frames (ORFs) on the right arm of chromosome XII of Saccharomyces cerevisiae. Using a PCR-based gene replacement strategy, we have systematically deleted individual ORFs and subjected the heterozygous diploids and haploid knockout strains to basic genetic and phenotypic characterization. Two ORFs, YLR127c and YLR129w, are essential for viability, whereas no growth phenotype could be detected following deletion of YLR124w, YLR125w, YLR126c or YLR128w. For each of the individual ORFs, a kanMX4 replacement cassette and the corresponding cognate wild-type gene were cloned into appropriate plasmids.

Identification of a pollen-specific sucrose transporter-like protein NtSUT3 from tobacco.

Pollen cells are symplasmically isolated during maturation and germination. Pollen therefore needs to take up nutrients via membrane carriers. Physiological measurements on pollen indicate sucrose transport in the pollen tube. A cDNA encoding a pollen-specific sucrose transporter-like protein NtSUT3 was isolated from a tobacco pollen cDNA library. NtSUT3 expression is detected only in pollen and is restricted to late pollen development, pollen germination and pollen tube growth. Altogether these data indicate that pollen is supplied not only with glucose, but also with sucrose through a specific sucrose transporter. The respective contribution of each transport pathway may change during pollen tube growth.

Deletion analysis of yeast Sec65p reveals a central domain that is sufficient for function in vivo.

The Saccharomyces cerevisiae SEC65 gene encodes a 32 kDa subunit of yeast signal recognition particle that is homologous to human SRP19. Sequence comparisons suggest that the yeast protein comprises three distinct domains. The central domain (residues 98-171) exhibits substantial sequence similarity to the 144 residue SRP19. In contrast, the N-terminal and C-terminal domains (residues 1-97 and 172-273 respectively) share no similarity to SRP19, with the exception of a cluster of positively charged residues at the extreme C-terminus of both proteins. Here, we report the cloning of a Sec65p homologue from the yeast Candida albicans that shares the same extended domain structure as its S. cerevisiae counterpart. This conservation of sequence is reflected at the functional level, as the C. albicans gene can complement the conditional lethal sec65-1 mutation in S. cerevisiae. In order to examine the role of the N- and C- terminal domains in Sec65p function, we have engineered truncation mutants of S. cerevisiae SEC65 and tested these for complementing activity in vivo and for SRP integrity in vitro. These studies indicate that a minimal Sec65p comprising residues 76-209, which includes the entire central SRP19-like domain, is sufficient for SRP function in yeast.

Protein translocation across the membrane of the endoplasmic reticulum.

Eukaryotic cells are characterized by the existence of membrane-bound subcellular compartments which perform a variety of specialized functions. Proteins destined for these compartments begin their synthesis in the cytosol and must be subsequently targeted to their functional compartment by specific signal sequences present in the newly synthesized polypeptide chain. The translocation of preproteins across biological membranes is a fundamental process of intracellular trafficking and organelle biogenesis. Entry into the secretory pathway occurs by translocation of proteins into or across the membrane of the endoplasmic reticulum (ER). This process involves two distinct steps which are dependent on the orchestrated action of several proteins. The initial step of targeting involves recognition of the signal sequence and delivery of the protein precursor to the ER in a translocation competent conformation. The subsequent translocation event is characterized by interaction of the preprotein with the translocation channel followed by unidirectional movement across the lipid bilayer of the ER membrane into the lumenal space. The study of the mechanism of the translocation process is one of the most intriguing and rapidly advancing areas in cell biology. Here we review recent findings in both the yeast Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process.

Isolation and sequence of HSP30, a yeast heat-shock gene coding for a hydrophobic membrane protein.

Using differential hybridization, a gene preferentially expressed during entry into stationary phase has been isolated. Subsequent analysis indicated that this gene corresponds to a heat-shock gene. The nucleotide sequence has been determined. It revealed a 332 amino-acid protein. No similarities to any previously known protein have been noted. The protein is very hydrophobic and is predicted to have a membraneous localisation. In agreement with this hypothesis, the analysis of membrane proteins from stationary-phase cells showed that a strain carrying this gene on a multicopy vector overproduces a protein of 30 kDa. This protein was recognized by antibodies directed against the N-terminal portion of the gene product. Considering its induction in response to heat shock and the apparent molecular weight of its product, this gene was designated HSP30.

Induction of a heat-shock-type response in Saccharomyces cerevisiae following glucose limitation.

The protein pattern of yeast cells which have arrested proliferation in response to glucose exhaustion is drastically different from that of exponentially growing cells (Boucherie, 1985). In this study, we used two-dimensional gel electrophoresis to characterize the protein events responsible for these alterations. We found that the induction of heat-shock proteins is one of the major events responsible for these changes. This induction accounts for the synthesis of 18 of the 35 novel polypeptides observed in glucose-limited cells. It was shown to occur in combination with two other protein events: the derepression of carbon catabolite repressed proteins, which accounts for the synthesis of the other novel polypeptides, and an arrest of the synthesis of almost all the proteins present in exponentially growing cells. The time course of each of these events was determined by carrying out a detailed analysis of the pattern of proteins synthesized at various stages of a culture exhausting its glucose supply, and by the measurement of the rate of synthesis of individual polypeptides. The results showed in particular that the synthesis of most of the heat-shock proteins synthesized in glucose-limited cells was induced closely before glucose exhaustion, and that this synthesis was transient, climaxing by the time glucose was exhausted. Under the culture condition investigated, the entry into stationary phase associated with glucose limitation began several hours before glucose exhaustion. It was thus concluded that the observed induction of heat-shock proteins is directly related to the nutritional limitation and is independent from the arrest of cell proliferation.

Functional analysis of the yeast genome: use of two-dimensional gel electrophoresis to detect genes in randomly cloned DNA sequences.

Genes are overexpressed when present in yeast cells on multicopy plasmids. Taking advantage of the protein amplification which results from this overexpression, a method has been developed for large scale detection of yeast genes on randomly cloned DNA sequences. It is based on the analysis, by two-dimensional gel electrophoresis, of the proteins from yeast cells transformed with a yeast genomic DNA library constructed in a multicopy vector. We demonstrate here the applicability of this method for exploring the yeast genome. In addition, we report results which suggest that this method may also be useful for detecting regulatory genes.