Proteasome‐ and SCF‐dependent degradation of yeast adenine deaminase upon transition from proliferation to quiescence requires a new F‐box protein named Saf1p

Abstract

SummaryIn response to nutrient limitation, Saccharomyces cerevisiae cells enter into a non-proliferating state termed quiescence. This transition is associated with profound changes in gene expression patterns. The adenine deaminase encoding gene AAH1 is among the most precociously and tightly downregulated gene upon entry into quiescence. We show that AAH1 downregulation is not specifically due to glucose exhaustion but is a more general response to nutrient limitation. We also found that Aah1p level is tightly correlated to RAS activity indicating thus an important role for the protein kinase A pathway in this regulation process. We have isolated three deletion mutants, srb10 , srb11 and saf1 ( ybr280c ) affecting AAH1 expression during post-diauxic growth and in early stationary phase. We show that the Srb10p cyclin-dependent kinase and its cyclin, Srb11p, regulate AAH1 expression at the transcriptional level. By contrast, Saf1p, a previously uncharacterized F-box protein, acts at a post-transcriptional level by promoting degradation of Aah1p. This post-transcriptional regulation is abolished by mutations affecting the proteasome or constant subunits of the SCF (Skp1-Cullin-F-box) complex. We propose that Saf1p targets Aah1p for proteasome-dependent degradation upon entry into quiescence. This work provides the first direct evidence for active degradation of proteins in quiescent yeast cells.

Skp1-Cullin-F-box-dependent Degradation of Aah1p Requires Its Interaction with the F-box Protein Saf1p

“…2, A and B), whereas deletion of SAF1 had no effect on Aah1p during exponential growth ( Fig. 2A) (9). This effect was confirmed by expressing Saf1p under control of a strong galactose-inducible promoter.…”

“…Transformants were then replicaplated in duplicate on SDcasaWA solid medium. One plate was conserved, and the other was tested for ␤-galactosidase activity by "agar overlay" as described previously (9). Plasmids from yeast colonies showing a high ␤-galactosidase activity were extracted and further characterized.…”

“…Colonies were then grown for 2 days on SDcasaWA medium, and ␤-galactosidase activity was revealed by agar overlay as described in Ref. 9. Putative mutants showing a blue color, revealing a high expression of AAH1-lacZ in stationary phase, were subcloned and assayed for ␤-galactosidase activity.…”

“…Several lines of argument support this idea. First, Saf1p is involved in the degradation of adenine deaminase (Aah1p), which occurs upon transition from proliferation to quiescence and which also depends on the E2 Cdc34p and core subunits of the SCF (Skp1p and Hrt1p) (9). Second, Saf1p was copurified with Cdc34p, Skp1p, and Cdc53p in three independent interactome analyses (13)(14)(15).…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…A genetic screen for mutants abolishing this regulation revealed that transcriptional regulation involves the kinase Ssn3 and its cyclin Ssn8 (Escusa et al 2006), which together are involved in phosphorylation of the RNA polymerase II C-terminal domain. The post-transcriptional regulation occurs through degradation of Aah1 by the proteasome and is mediated by a SCF complex involving the F-box protein Saf1, which is itself upregulated in stationary phase (Escusa et al 2006(Escusa et al , 2007. However, a saf1 mutant can enter and exit stationary phase normally, and the role of nucleotide synthesis in stationary-phase establishment is still unclear.…”