Pheromone induction promotes Ste11 degradation through a MAPK feedback and ubiquitin-dependent mechanism.

Ste11 is the mitogen-activated protein kinase (MAPK) kinase kinase in the MAPK cascades that mediate mating, high osmolarity glycerol, and filamentous growth responses in Saccharomyces cerevisiae. We show stimulation of the mating pathway by pheromone promotes an accelerated turnover of Ste11 through a MAPK feedback and ubiquitin-dependent mechanism. This degradation is pathway specific, because Ste11 is stable during activation of the high osmolarity glycerol pathway. Because the steady-state amount of Ste11 does not change significantly during pheromone induction, we infer that maintenance of MAPK activation involves repeated cycles in which naive Ste11 is activated and then targeted for degradation. This model predicts that elimination of active Ste11 would rapidly curtail MAPK activation upon attenuation of the upstream signal. This prediction is confirmed by the finding that blocking ubiquitin-dependent Ste11 degradation during pheromone induction abolishes the characteristic attenuation profile for MAPK activation.

Cooperative binding interactions required for function of the Ty1 sterile responsive element.

The Ste12p transcription factor controls the expression of Ty1 transposable element insertion mutations and genes whose products are required for mating in Saccharomyces cerevisiae. The binding site for Ste12p is a consensus DNA sequence known as a pheromone response element (PRE). Upstream activating sequences (UASs) derived from known Ste12p-dependent genes have previously been characterized to require either multiple PREs or a single PRE coupled to a binding site for a second protein. The Ste12p-dependent UAS from Ty1, called a sterile response element (SRE), is of the second type and is comprised of a PRE and an adjacent TEA (TEF-1, Tec1, and AbaA motif) DNA consensus sequence (TCS). In this report, we show by UV cross-linking analysis that two proteins, Ste12p and a protein with an apparent size of 72 kDa, directly contact the Ty1 SRE. Other experiments show that Tec1p is required for formation of the Ty1 SRE protein-DNA complex and is physically present in the complex. These results establish a direct role for Tec1p in the Ty1 SRE and yet another set of combinatorial interactions that achieve a qualitatively distinct mode of transcriptional regulation with Ste12p.

Regulation of the Dictyostelium cAMP-induced, prestalk-specific DdrasD gene: identification of cis-acting elements.

We have shown previously that expression of the Dictyostelium ras gene DdrasD (previously denoted Ddras) is induced during multicellular development and in single-cell shaking culture in response to cAMP (1). Analysis of transformants carrying DdrasD/lacZ reporter constructs showed DdrasD expression to be prestalk-specific (2). The gene is transcribed from three start sites with transcription from the distal site producing an approximately 1.2 kb transcript, which is expressed at low levels in growing cells and is subsequently induced late in aggregation. This promoter is also induced to high levels by cAMP. Transcription from the two more proximal sites is coregulated and is induced during development, resulting in approximately 1.0 kb transcripts. In this study, we examine cis-acting regions required for proper regulation of DdrasD expression using a DdrasD/beta-glucuronidase reporter gene construct. We have identified distinct sequence elements required for developmental and vegetative expression of DdrasD. A domain containing a CA repeat, similar to ones found in other late, cAMP-induced Dictyostelium genes, is required for cAMP-induced and developmental expression.

cAMP and cell sorting control the spatial expression of a developmentally essential cell-type-specific ras gene in Dictyostelium.

The Dictyostelium ras gene (Dd-ras) is expressed at a low level in vegetative cells, is not expressed between the onset of development and aggregation, and is then re-expressed in the multicellular aggregate stages from the distal, now cAMP-responsive, promoter and from two more proximal promoters. Expression of activated Dd-ras (G12----T12) (Reymond et al. 1986) results in an abnormal developmental phenotype with the formation of aggregates having multiple tips and an inhibition of further development. In this report we investigate the spatial expression of Dd-ras by fusing the 5'-flanking region to the Escherichia coli lacZ gene and by staining aggregates for beta-galactosidase (beta-gal) activity. We show that fusions using 5'-flanking sequences that include all promoters are expressed in approximately 10-20% of the cells randomly scattered within the early aggregate. Our data indicate that these beta-gal-expressing cells migrate to newly formed tips of aggregates and localize in the region that becomes the prestalk zone. Staining is also seen in the very posterior of the organism. The anterior staining appears to be specific for the prestalk A population, and beta-gal activity is subsequently present in stalk cells as developmental proceeds. When only the two more proximal promoters are used to drive lacZ expression, localized staining is seen in the anterior prestalk region, although it is weaker than with the construct carrying all promoters. Moreover, staining is not seen in the posterior domain in the first finger stage, suggesting differences in the spatial expression from the different promoters. Staining is also observed in some cells within the prespore region, which could be anterior-like cells. The pattern of Dd-ras/lacZ staining during tip formation suggests a directed, spiral pattern of cell migration, possibly in response to the proposed spiral gradient of cAMP within the developing aggregate. The pattern of Dd-ras is consistent with the abnormal developmental phenotype caused by expressing an activated Dd-ras Thr12 gene and suggests an essential role for Dd-ras in controlling spatial differentiation.