The highly conserved, coregulated SNO and SNZ gene families in Saccharomyces cerevisiae respond to nutrient limitation.

SNZ1, a member of a highly conserved gene family, was first identified through studies of proteins synthesized in stationary-phase yeast cells. There are three SNZ genes in Saccharomyces cerevisiae, each of which has another highly conserved gene, named SNO (SNZ proximal open reading frame), upstream. The DNA sequences and relative positions of SNZ and SNO genes have been phylogenetically conserved. This report details studies of the expression of the SNZ-SNO gene pairs under various conditions and phenotypic analysis of snz-sno mutants. An analysis of total RNA was used to determine that adjacent SNZ-SNO gene pairs are coregulated. SNZ2/3 and SNO2/3 mRNAs are induced prior to the diauxic shift and decrease in abundance during the postdiauxic phase, when SNZ1 and SNO1 are induced. In snz2 snz3 mutants, SNZ1 mRNA is induced prior to the diauxic shift, when SNZ2/3 mRNAs are normally induced. Under nitrogen-limiting conditions, SNZ1 mRNAs accumulate in tryptophan, adenine, and uracil auxotrophs but not in prototrophic strains, indicating that induction occurs in response to the limitation of specific nutrients. Strains carrying deletions in all SNZ-SNO gene pairs are viable, but snz1 and sno1 mutants are sensitive to 6-azauracil (6-AU), an inhibitor of purine and pyrimidine biosynthetic enzymes, and methylene blue, a producer of singlet oxygen. The conservation of sequence and chromosomal position, the coregulation and pattern of expression of SNZ1 and SNO1 genes, and the sensitivity of snz1 and sno1 mutants to 6-AU support the hypothesis that the associated proteins are part of an ancient response to nutrient limitation.

Yeast bcy1 mutants with stationary phase-specific defects.

Entry into the stationary phase requires the yeast BCY1 gene, which encodes the regulatory subunit of the cAMP-dependent protein kinase (cAPK). New bcy1 mutants, constructed by in vitro mutagenesis of the 3'-region encoding the cAMP-binding domains, were classified as early or late-acting mutants based on viability studies. The late-acting bcy1 mutants accumulated fewer stationary phase-specific Bcy1p isoforms and had decreased cAPK activity. This late-acting class is novel and dies after 7 days in culture, later than two previously reported stationary phase mutants, ubi4 and ard1.

A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.

Protein synthesis in long-term stationary-phase cultures of Saccharomyces cerevisiae.

We are interested in characterizing the process of entry into and the maintenance of the stationary phase. To identify proteins that are induced during growth to stationary phase, we examined protein synthesis in long-term stationary-phase cultures using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Although the total rate of protein synthesis declined when growth ceased after the postdiauxic phase, the pattern of proteins synthesized remained similar throughout the experimental period (28 days), except at the diauxic shift. At the diauxic shift most proteins detectable by 2D-PAGE undergo a transient reduction in their relative rate of synthesis that ends when cells resume growth during the postdiauxic phase. We conclude from this that the transient repression of protein synthesis at the diauxic shift is not directly associated with stationary-phase arrest. A number of proteins that are synthesized after exponential phase have been identified by 2D-PAGE. These proteins could be divided into three temporal classes depending upon when their synthesis became detectable. One postexponential protein, designated p35, was induced later than all other proteins, and its relative rate of synthesis increased throughout stationary phase. Unlike most postexponential proteins, p35 was not regulated by heat shock or glucose repression. We also observed that a direct correlation between steady-state mRNA accumulation and protein synthesis for another postexponential protein (Ssa3p) or two closely related constitutive proteins (Ssa1p and Ssa2p) did not exist. We conclude from this result that synthesis of proteins in stationary phase is regulated by mechanisms other than the control of steady-state mRNA accumulation.

AppppA-binding protein E89 is the Escherichia coli heat shock protein ClpB.

Dinucleotide AppppA (5',5'''-P1,P4-diadenosine tetraphosphate) is rapidly synthesized in Escherichia coli cells during heat shock. apaH mutants lack AppppN hydrolase activity and, therefore, contain constitutively levels of AppppA, which affect several cellular processes. However, the precise role of AppppA remains undetermined. Photo-crosslinking experiments with radioactively labelled azido-AppppA have shown that a number of proteins, including heat shock proteins DnaK and GroEL, specifically bind to AppppA. Several other unidentified proteins (C40, C45, and E89) also bind strongly to AppppA. In this work, we have identified the AppppA-binding protein E89 as heat shock protein ClpB. In addition, since ClpB belongs to a family of proteins implicated in proteolysis, we have examined the effects of apaH mutants on protein degradation. Constitutively elevated levels of AppppA stimulate lon-independent proteolysis only in heat-shocked cells. We also show that overproduction of ClpB from a plasmid rescues apaH mutants from sensitivity to killing by heat.