The dual-specificity protein phosphatase Yvh1p regulates sporulation, growth, and glycogen accumulation independently of catalytic activity in Saccharomyces cerevisiae via the cyclic AMP-dependent protein kinase cascade

Abstract

Yvh1p, a dual-specific protein phosphatase induced specifically by nitrogen starvation, regulates cell growth as well as initiation and completion of sporulation. We demonstrate that yvh1 disruption mutants are also unable to accumulate glycogen in stationary phase. A catalytically inactive variant of yvh1 (C117S) and a DNA fragment encoding only the Yvh1p C-terminal 159 amino acids (which completely lacks the phosphatase domain) complement all three phenotypes as well as the wild-type allele; no complementation occurs with a fragment encoding only the C-terminal 74 amino acids. These observations argue that phosphatase activity is not required for the Yvh1p functions we measured. Mutations which decrease endogenous cyclic AMP (cAMP) levels partially suppress the sporulation and glycogen accumulation defects. In addition, reporter gene expression supported by a DRR2 promoter fragment, containing two stress response elements known to respond to cAMP-protein kinase A, decreases in a yvh1 disruption mutant. Therefore, our results identify three cellular processes that both require Yvh1p and respond to alterations in cAMP, and they lead us to suggest that Yvh1p may be a participant in and/or a contributor to regulation of the cAMP-dependent protein kinase cascade. The fact that decreasing the levels of cAMP alleviates the need for Yvh1p function supports this suggestion.

FIG. 1

Glycogen accumulation in stationary-phase cells. Wild-type (GYC86) (A), yvh1Δ (HPY120) (B), pho85Δ (GYC134) (C), and yvh1Δ pho85Δ (GYC135) (D) cultures were grown in YEPD medium, and aliquots were withdrawn at the A₆₀₀ indicated on the ordinate and quantified for glycogen as described in Materials and Methods. Each value represents the average of two determinations per aliquot. (E) Acid phosphatase assay. Wild-type (GYC86), yvh1Δ (HPY120), pho85Δ (GYC134), and yvh1Δ pho85Δ (GYC134) cells were streaked on high Pi X-P synthetic complete plates and incubated at 30°C. The appearance of blue color indicates acid phosphatase expression.

FIG. 2

Effects of progressive C-terminal yvh1 deletions on their ability to complement the yvh1 null mutation in strain HPY120. (A) Normalized glycogen accumulation 72 h after inoculation of strain HPY120, transformed with plasmids indicated on the abscissa, into synthetic complete (SC) Ura⁻ medium. (B) Spore maturation (dityrosine fluorescence). (C and D) Growth on SC Ura⁻ plates. All transformants were streaked onto the respective plate and incubated at 30°C.

FIG. 3

Phosphatase activity of wild-type and catalytically inactive alleles of YVH1. (A) pNPP activities of purified wild-type (pAB36) and catalytically inactive (pAB37) Yvh1p-GST fusion proteins. (B) Silver-stained polyacrylamide gel electrophoresis (PAGE) gels of the proteins used in panel A. (C) Western analyses of various YVH1 alleles fused to GST.

FIG. 4

Abilities of various GST tagged alleles of YVH1 to suppress the slow-growth defect of yvh1 strain HPY120 on glucose (A) and raffinose (B) plates. (C) Growth phenotypes of various strains used in this work, including the integrated catalytically inactive allele of YVH1, on YEPD plates. All strains and transformants were grown at 30°C.

FIG. 5

Abilities of various GST tagged alleles of YVH1 to suppress the glycogen accumulation and spore maturation defects of yvh1 strain HPY120. (A and B) Normalized glycogen accumulation of transformants 72 h postinoculation into synthetic complete (SC) Ura⁻ medium with glucose (A) or raffinose (B) as the carbon source. (C) Spore maturation (dityrosine fluorescence) assays using the standard spore maturation protocol described in Materials and Methods.

FIG. 6

Protein phosphatase activity is not required for glycogen accumulation or spore maturation. (A) Normalized glycogen content for wild-type (GYC86), yvh1Δ (HPY120), mck1Δ (YSB40), pho85Δ (GYC134), and HAyvh1C117S (GYC136) strains 72 h postinoculation. (B) Spore maturation phenotypes of wild-type (GYC86), yvh1Δ (HPY120), HAYVH1 (YSB32), HAyvh1C117S (GYC136), and mck1Δ (YSB40) strains under standard conditions.

FIG. 7

Conditional expression of genes responsible for cAMP turnover, PDE1 (pAB112) and PDE2 (pAB113), can partially suppress the glycogen accumulation and spore maturation defects of yvh1Δ in an expression-dependent manner. (A and B) Normalized glycogen accumulation 72 h postinoculation in glucose (A) or raffinose (B) medium. (C and D) Spore maturation phenotypes of the same transformants under the standard dityrosine protocol (C) and the unconventional protocol (D) as described in Materials and Methods.

FIG. 8

Conditional expression of PDE1 and PDE2 does not suppress the slow-growth phenotype of yvh1 mutants. Strain HPY120 was transformed with GST fused to one of the following genes: none (pEGKG; vector), YVH1 (pAB36), yvh1C117S (pAB37), GSY2 (pAB111), PDE1 (pAB112), and PDE2 (pAB113). Transformants were streaked onto minimal glucose (A) or raffinose (B) medium and grown at 30°C; these conditions yield low and moderate levels of fusion gene expression. Full induction of the GAL1,10 promoter, driving the above gene expression, is highly detrimental to growth of both the yvh1 (HPY120) and wild-type (GYC86) strains transformed with pAB112 and pAB113.

FIG. 9

Deletion of YVH1 results in decreased induction of transcription mediated by a multi-STRE. Wild-type (GYC86) and yvh1 (HPY120) strains were transformed with the lacZ reporter plasmid pNG15 or the same plasmid containing the DDR2 promoter element (pAB60). Transformants were grown at 22°C (non-stress condition) and then shifted to 40°C for 1 h (stress condition) before expression was assayed. Values represent the average of four independent transformants assayed in duplicate.

FIG. 10

Model accounting for physiological functions in which YVH1p participates.