Protein-protein interactions in the yeast

Abstract

The two-hybrid system for the identification of protein-protein interactions was used to screen for proteins that interact in vivo with the Saccharomyces cerevisiae Pkc1 protein, a homolog of mammalian protein kinase C. Four positive clones were isolated that encoded portions of the protein kinase Mkk1, which acts downstream of Pkc1p in the PKC1-mediated signalling pathway. Subsequently, Pkc1p and the other PKC1 pathway components encoding members of a MAP kinase cascade, Bck1p (a MEKK), Mkk1p, Mkk2p (two functionally homologous MEKs), and Mpk1p (a MAP kinase), were tested pairwise for interaction in the two-hybrid assay. Pkc1p interacted specifically with small N-terminal deletions of Mkk1p, and no interaction between Pkc1p and any of the other known pathway components could be detected. Interaction between Pkc1p and Mkk1p, however, was found to be independent of Mkk1p kinase activity. Bck1p was also found to interact with Mkk1p and Mkk2p, and the interaction required only the predicted C-terminal catalytic domain of Mkk1p. Furthermore, we detected protein-protein interactions between two Bck1p molecules via their N-terminal regions. Finally, Mkk2p and Mpk1p also interacted in the two-hybrid assay. These results suggest that the members of the PKC1-mediated MAP kinase cascade form a complex in vivo and that Pkc1p is capable of directly interacting with at least one component of this pathway.

Rom2p, the Rho1 GTP/GDP Exchange Factor of Saccharomyces cerevisiae, Can Mediate Stress Responses via the Ras-cAMP Pathway

“…Furthermore, the expression of a constitutively active allele of RHO1 had no effect on resistance to caffeine, indicating that the response to this compound is probably mediated through another pathway. This extends the potential involvement of the Rho1-PKC pathway in stress responses well beyond the previously observed heat shock (33)(34)(35) and hypo-osmotic stresses (35)(36)(37). Recently, it has been shown that the PKC-activated MAP kinase Mpk1p is rapidly and multiply phosphorylated following the exposure of cells to low concentrations of linoleate hydroperoxide (59); hence, under one condition at least the cell integrity pathway MAP kinase is activated by one form of oxidative stress.…”

“…The Rho1-protein kinase C (Rho1-PKC) pathway, known as the cellular integrity pathway for its role in cell wall biosynthesis and mating (30 -32), can also mediate stress responses to temperature shift (33)(34)(35) and changes in osmolarity (35)(36)(37). The Rho1-PKC pathway is connected to the TOR2 pathway via Rom2p (38,39), which is the guanine nucleotide exchange factor for Rho1p (40).…”

Retrophosphorylation of Mkk1 and Mkk2 MAPKKs by the Slt2 MAPK in the Yeast Cell Integrity Pathway

“…This domain obeys to the consensus (K/R) 2-3 X 1-6 (L/I)X(L/I) in mammalian MAPK pathways (45,46) as well as in the yeast mating pheromone pathway (30). Although Mkk1 and Mkk2 are known to interact with Slt2 via their N-terminal region (36,47), a canonical D-domain has not been mapped in these MAPKKs. We aligned the N-terminal region of Mkk1 and Mkk2 from S. cerevisiae as well as their orthologs in other fungi in FIGURE 4.…”

Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two‐hybrid analysis and functional complementation tests of yeast mutants

“…Recently, yeast 2‐hybrid analyses have revealed physical interactions among components of animal and yeast MAPK cascades whose functional interactions had been identified by biochemical and genetic analyses [11–16]. In the sexual response pathway of Schizosaccharomyces pombe , byr1 (a MAPKK) interacts with spk1 (a MAPK) [15].…”