The multicopy suppressors of the snf1 defect, Msn2p and Msn4p transcription factors (Msn2/4p), activate genes through the stress-responsive cis element (CCCCT) in response to various stresses. This cis element is also the target for repression by the cyclic AMP (cAMP)-signaling pathway. We analyzed the two-dimensional gel electrophoresis pattern of protein synthesis of themsn2 msn4 double mutant and compared it with that of the wild-type strain during exponential growth phase and at the diauxic transition. Thirty-nine gene products (including those ofALD3, GDH3, GLK1, GPP2,HSP104, HXK1, PGM2,SOD2, SSA3, SSA4, TKL2,TPS1, and YBR149W) are dependent upon Msn2/4p for their induction at the diauxic transition. The expression of all these genes is repressed by cAMP. Thirty other genes identified during this study are still inducible in the mutant. A subset of these genes were found to be superinduced at the diauxic transition, and others were subject to cAMP repression (including ACH1,ADH2, ALD6, ATP2, GPD1,ICL1, and KGD2). We conclude from this analysis that Msn2/4p control a large number of genes induced at the diauxic transition but that other, as-yet-uncharacterized regulators, also contribute to this response. In addition, we show here that cAMP repression applies to both Msn2/4p-dependent and -independent control of gene expression at the diauxic shift. Furthermore, the fact that all the Msn2/4p gene targets are subject to cAMP repression suggests that these regulators could be targets for the cAMP-signaling pathway.

SummaryThe heat shock transcription factor Hsf1p and the stress-responsive transcription factors Msn2p and Msn4p are activated by heat shock in the yeast Saccharomyces cerevisiae. Their respective contributions to heat shock protein induction have been analysed by comparison of mutants and wild-type strains using [ 35 S]-methionine labelling and two-dimensional gel electrophoresis. Among 52 proteins induced by a shift from 258C to 388C, half of them were found to be dependent upon Msn2p and/or Msn4p (including mostly antioxidants and enzymes involved in carbon metabolism), while the other half (including mostly chaperones and associated proteins) were dependent upon Hsf1p. The two sets of proteins overlapped only slightly. Three proteins were induced independently of these transcription factors, suggesting the involvement of other transcription factor(s). The Ras/cAMP/PKA signalling pathway cAMP had a negative effect on the induction of the Msn2p/Msn4p regulon, but did not affect the Hsf1p regulon. Thus, the two types of transcription factor are regulated differently and control two sets of functionally distinct proteins, suggesting two different physiological roles in the heat shock cellular response.

Nascent proteins synthesized by membrane-bound ribosomes are translocated across the ER 1 membrane and acquire carbohydrate chains on specific serine, threonine, and asparagine residues. These glycoproteins then proceed through the Golgi complex where their oligosaccharides are further modified. The O-modified proteins of Saccharomyces cerevisiae possess a linear carbohydrate chain of up to 5 mannose residues (1,2). O-Glycosylation is initiated in the ER with dolichyl phosphate-D-mannose as the immediate sugar donor for the mannosyl residue transferred to the hydroxy-amino acids serine and threonine (2, 3). GDP-Man is utilized as the sugar donor in the subsequent elongation of O-linked carbohydrate chains resulting in a linear glycan in which the second and third mannoses possess ␣1,2-linkages, whereas the terminal fourth and fifth mannosyl residues have ␣1,3-linkages (1, 2).Yeast N-linked modified proteins can acquire two different types of glycan chains after the addition of a Man 8 GlcNAc 2 core in the endoplasmic reticulum, a process common to the majority of eukaryotes (1, 2). This primary oligosaccharide can undergo maturation in the Golgi resulting in a Man 8 -13 GlcNAc 2 carbohydrate structure, or it may be extended by an outer chain of variable size (up to 200 mannose residues) that is made up of a backbone of ␣1,6-mannosyl residues to which are attached branched ␣1,2-and ␣1,3-mannosyl side chains.Some of the enzymes involved in the elaboration of O-linked oligosaccharides and in the synthesis of N-linked outer chains have been identified, and their structural genes have been isolated. At least four different genes of the seven-membered PMT1-7 gene family encoding dolichyl phosphate-D-mannose: protein O-D-mannosyltransferases are responsible for initiating the O-linked glycans (4). Protein O-glycosylation is essential for cell function because mutants of S. cerevisiae lacking different combinations of three of the PMT genes are not viable (4). The OCH1 gene is responsible for adding the first ␣1,6-mannose residue involved in initiating N-linked outer chain elaboration (5, 6). Two enzymes in particular have been shown to participate in both O-and N-linked glycosylation. The KRE2/ MNT1 gene encodes a medial Golgi ␣1,2-mannosyltransferase required for the addition of the third mannose residue on O-linked chains (7,8) and is also implicated in N-linked outer chain oligosaccharide synthesis (9, 10). The O-linked trisaccharide can be further extended by one or two ␣1,3-linked mannoses. The fourth mannose residue is added by the Golgi localized ␣1,3-mannosyltransferase Mnn1p, which also terminally mannosylates core and outer chain modified N-linked glycans (8,(11)(12)(13).To further characterize protein glycosylation in yeast and identify some of the responsible glycosyltransferases, we have studied a gene family encoding proteins that are highly homologous to Kre2p/Mnt1p. This nine-membered KTR mannosyltransferase gene family contains the KRE2/MNT1, YUR1, KTR1, KTR2, KTR3, KTR4, KTR5, KTR6,. As with other kno...

DNA–lacZ fusion libraries of yeast Saccharomyces cerevisiae were used to select genes coordinately regulated by the Ras–cAMP–cAPK signalling pathway. Sixteen new genes (AGP1, APE2, APE3, FPS1, GUT2, MDH2, PLB2, PYK2, RNR3, SUR1, UGA1, YHR033w, YBR006w, YHR143w, YMR086w and YOR173w) were found to be repressed by cAMP. Most of these genes encode for metabolic enzymes and are induced by nutritional limitations. These common properties suggest a role of this pathway in the metabolic adjustment of the cell to nutritional variations. The induction of 10 of these genes is reduced in the msn2,msn4 double mutant, which emphasizes the role of the Msn2/4p transcriptional activators in mediating the Ras–cAMP–cAPK signalling pathway. The Msn2p/Msn4p‐independent expression of the six other genes suggests the existence of other regulatory systems under the control of this pathway. Copyright © 1999 John Wiley & Sons, Ltd.

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