Krh1p and Krh2p act downstream of the Gpa2p Gα subunit to negatively regulate haploid invasive growth

Abstract

The yeast Gα subunit Gpa2p and its coupled receptor Gpr1p function in a signaling pathway that is required for the transition to pseudohyphal and invasive growth. A two-hybrid screen using a constitutively active allele of GPA2 identified the KRH1 gene as encoding a potential binding partner of Gpa2p. Strains containing deletions of KRH1 and its homolog KRH2 were hyper-invasive and displayed a high level of expression of FLO11, a gene involved in pseudohyphal and invasive growth. Therefore, KRH1 and KRH2 encode negative regulators of the invasive growth pathway. Cells containing krh1Δ krh2Δ mutations also displayed increased sensitivity to heat shock and decreased sporulation efficiency, indicating that Krh1p and Krh2p regulate multiple processes controlled by the cAMP/PKA pathway. The krh1Δ krh2Δ mutations suppressed the effect of a gpa2Δ mutation on FLO11 expression and eliminated the effect of a constitutively active GPA2 allele on induction of FLO11 and heat shock sensitivity, suggesting that Krh1p and Krh2p act downstream of Gpa2p. The Sch9p kinase was not required for the signal generated by deletion of KRH1 and KRH2; however, the cAMP-dependent kinase Tpk2p was required for generation of this signal. These results support a model in which activation of Gpa2p relieves the inhibition exerted by Krh1p and Krh2p on components of the cAMP/PKA signaling pathway.

Kelch-repeat proteins interacting with the G α protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast

“…To investigate the functional importance of these proteins in Gpa2 signaling, we deleted KRH1 and KRH2 and determined typical PKA-associated parameters when cells approach stationary phase. Consistent with the hyperfilamentous phenotype of mutants lacking Krh1͞2 (22,23), indicative of high PKA activity, absence of the kelch-repeat proteins resulted in constitutively low levels of the reserve carbohydrates trehalose and glycogen and low expression of HSP12 ( Fig. 1; glycogen data not shown).…”

“…In an in vitro binding assay, Krh1 and Krh2 were found to bind to both GDP-and GTP-loaded Gpa2, but they bound with the highest affinity to GDP-Gpa2 (22). On the other hand, in the yeast two-hybrid system, a constitutively active Gpa2 mutant (Gpa2 R273A ) was reported to associate better with the C-terminal half of Krh1 than WT Gpa2 (23). Furthermore, adenylate cyclase (Cyr1) has been suggested to be an effector of Gpa2, based on genetic data (14).…”

“…For this reason, we have adopted the neutral terminology ''Krh'' (kelch repeat homologue, as in ref. 23) throughout this article rather than the suggestive ''Gpb'' (G protein ␤; ref. 22).…”

“…Recently, two related interaction partners of Gpa2 have been described: Krh1͞Gpb2 and Krh2͞Gpb1 (22,23). Henceforward, we refer to these proteins as Krh (kelch repeat homologue) 1 and Krh2, as argued in the first paragraph of Results.…”

“…Recently, the same researchers reported that Krh1 impairs efficient coupling of Gpa2 to its cognate receptor, Gpr1, by binding to the extended N terminus of Gpa2 (24). However, absence of Gpa2 does not suppress the high expression of FLO11 or the hyperfilamentation in a double krh1͞2⌬ mutant, suggesting that these kelch-repeat proteins largely act downstream or independently of Gpa2 (23). On the other hand, deletion of TPK2, encoding the PKA catalytic subunit that is specifically required for filamentous growth (5,25), largely abrogates the hyperinvasive phenotype of the krh1͞2⌬ mutant (22,23), arguing that Krh1 and Krh2 function upstream of PKA.…”