Irreversible inactivation of Saccharomyces cerevisiae fructose‐1,6‐bisphosphatase independent of protein phosphorylation at Ser¹¹

Abstract

The fructose-1,6-bisphosphatase gene was used with multicopy plasmids to study rapid reversible and irreversible inactivation after addition of glucose to derepressed Saccharomyces cerevisiae cells. Both inactivation systems could inactivate the enzyme, even if 20-fold over-expressed. The putative serine residue, at which fructose-1,6-bisphosphatase is phosphorylated, was changed to an alanine residue without notably affecting the catalytic activity. No rapid reversible inactivation was observed with the mutated enzyme. Nonetheless, the modified enzyme was still irreversibly inactivated, clearly demonstrating that phosphorylation is an independent regulatory circuit that reduces fructose-1,6-bisphosphatase activity within seconds. Furthermore, irreversible glucose inactivation was not triggered by phosphorylation of the enzyme.

Efficient transition to growth on fermentable carbon sources in Saccharomyces cerevisiae requires signaling through the Ras pathway

“…However, the reciprocal reaction is catalyzed by phosphofructokinase (PFK) during growth on a fermentable carbon source, and FBPase expression is repressed under these conditions. In the transition from non‐fermentative to fermentative growth, phosphorylation of FBPase by PKA inactivates it and targets it for Pep4‐mediated degradation in the vacuole (Pohlig and Holzer, 1985; Rose et al ., 1988; Chiang and Schekman, 1991; Huang and Chiang, 1997). To address whether the lack of a transient increase in cAMP in ras2 318S cells affects the activity of PKA, we examined the phosphorylation and decay of FBPase in cells following addition of glucose to glycerol‐grown cells.…”

“…PKA stimulates PFK indirectly by stimulating production of fructose‐2,6‐bisphosphate (F‐2,6‐BiP), an allosteric activator of PFK and inhibitor of FBPase. In addition, direct phosphorylation of FBPase by PKA inhibits its activity, increases its sensitivity to F‐2,6‐BiP and targets the protein for degradation by stimulating its vesicle‐mediated transport to the vacuole where it is proteolyzed in a Pep4‐dependent manner (Pohlig and Holzer, 1985; Rose et al ., 1988; Chiang and Schekman, 1991; Huang and Chiang, 1997). Thus, PKA can play a direct role in reconfiguring the primary carbon metabolic pathways in the cell.…”

Proteins of Newly Isolated Mutants and the Amino-terminal Proline Are Essential for Ubiquitin-Proteasome-catalyzed Catabolite Degradation of Fructose-1,6-bisphosphatase of Saccharomyces cerevisiae

“…Generation of Strains YMH1, YMH2, and YMH4 -The linearized FBPase-S11A fragment was obtained by HindIII, SalI cleavage of plasmid pRV44 (13). The cleaved SalI site was obtained from the flanking vector sequence.…”

“…pRV44 is a YEp24-based plasmid that contains the FBPase encoding sequence under the control of its native promotor (13). YEp112 is a 2-m-based S. cerevisiae-Escherichia coli shuttle vector that encodes a synthetic version of yeast ubiquitin (Ub) carrying an epitope from the hemagglutinin (ha) of influenza virus attached to the amino terminus of ubiquitin (haUb) under the control of the copper-inducible CUP1 promotor.…”

“…This modification was proposed to target the protein to the proteolytic machinery for degradation (7,8). However, site-directed mutagenesis of the serine to an alanine residue, by this preventing phosphorylation, showed no effect and disproved its importance for inactivation (13). Degradation of FBPase due to selective uptake into the vacuole and subsequent hydrolysis dependent on the vacuolar proteinase yscA has been reported (14).…”