Repressors and Upstream Repressing Sequences of the Stress-Regulated ENA1 Gene in Saccharomyces cerevisiae: bZIP Protein Sko1p Confers HOG-Dependent Osmotic Regulation

Abstract

The yeast ENA1/PMR2A gene encodes a cation extrusion ATPase in Saccharomyces cerevisiae which is essential for survival under salt stress conditions. One important mechanism of ENA1 transcriptional regulation is based on repression under normal growth conditions, which is relieved by either osmotic induction or glucose starvation. Analysis of the ENA1 promoter revealed a Mig1p-binding motif (؊533 to ؊544) which was characterized as an upstream repressing sequence (URS MIG-ENA1 ) regulated by carbon source. Its function was abolished in a mig1 mig2 double-deletion strain as well as in either ssn6 or tup1 single mutants. A second URS at ؊502 to ؊513 is responsible for transcriptional repression regulated by osmotic stress and is similar to mammalian cyclic AMP response elements (CREs) that are recognized by CREB proteins. This URS CRE-ENA1 element requires for its repression function the yeast CREB homolog Sko1p (Acr1p) as well as the integrity of the Ssn6p-Tup1p corepressor complex. When targeted to the GAL1 promoter by fusing with the Gal4p DNA-binding domain, Sko1p acts as an Ssn6/Tup1p-dependent repressor regulated by osmotic stress. A glutathione S-transferase-Sko1 fusion protein binds specifically to the URS CRE-ENA1 element. Furthermore, a hog1 mitogen-activated protein kinase deletion strain could not counteract repression on URS CRE-ENA1 during osmotic shock. The loss of SKO1 completely restored ENA1 expression in a hog1 mutant and partially suppressed the osmotic stress sensitivity, qualifying Sko1p as a downstream effector of the HOG pathway. Our results indicate that different signalling pathways (HOG osmotic pathway and glucose repression pathway) use distinct promoter elements of ENA1 (URS CRE-ENA1 and URS MIG-ENA1 ) via specific transcriptional repressors (Sko1p and Mig1/2p) and via the general Ssn6p-Tup1p complex. The physiological importance of the relief from repression during salt stress was also demonstrated by the increased tolerance of sko1 or ssn6 mutants to Na ؉ or Li ؉ stress.

MAP kinases as structural adaptors and enzymatic activators in transcription complexes

“…It regulates several transcription factors, each responsible for controlling expression of a subset of osmoresponsive genes. These are the Hot1p, Smp1p, Msn1p, Msn2p and Msn4p activators and the Sko1p repressor Martinez-Pastor et al, 1996;Proft and Serrano, 1999;Rep et al, 2000;Rep et al, 2001;Rep et al, 1999). We have known for some time that the nuclear retention of certain MAPKs upon stress requires the presence of their transcription factor substrates (Reiser et al, 1999a).…”

“…An ATF/CREB-family member, Sko1p, represses expression of a subset of osmoresponsive genes by recruiting the co-repressor complex Cyc8p-Tup1p (Garcia-Gimeno and Struhl, 2000;Proft and Serrano, 1999;Rep et al, 2001). Osmotic stress leads to binding of active Hog1p to Sko1p at these promoters and the subsequent phosphorylation of Sko1p by Hog1p Proft and Struhl, 2002).…”

Phenotypic effects of membrane protein overexpression in Saccharomyces cerevisiae

“…Notably, both components of the main K ϩ transport system (Trk1p and Trk2p) (27) the three major membrane transporters mediating Na ϩ export (Ena1p, Ena2p, and Ena5p) (28,29), and the previously identified Na ϩ tolerance protein Sna3p (30) scored among the most caffeine-tolerant overexpression strains. The Ena1p-induced caffeine tolerance may be related to the fact that PKA activity influences the nucleocytosolic localization of the basic leucine zipper repressor Sko1p (31) that, in turn, represses ENA1 expression by binding to the cyclicAMP response-like element in the ENA1 promoter (32).…”

A chromatin-mediated mechanism for specification of conditional transcription factor targets

“…Tup1 is a well-characterized repressor that does not bind DNA directly 15 , but is instead recruited to specific loci by other sequence-specific factors. Mig1, Nrg1, and Sko1 recruit Tup1 to the promoters of glucose-repressed genes, starch degrading genes, and osmotic stress inducible genes respectively [16][17][18] . We hypothesized that Tup1 and its recruiters negatively regulate Rap1 binding to low-glucose targets.…”