Activation Domain-dependent Monoubiquitylation of Gal4 Protein Is Essential for Promoter Binding in Vivo

Chase T Archer,Agnes Delahodde,Fernando Gonzalez,Stephen Albert Johnston,Thomas Kodadek

doi:10.1074/jbc.m801050200

Abstract

The Saccharomyces cerevisiae Gal4 protein is a paradigmatic transcriptional activator containing a C-terminal acidic activation domain (AD) of 34 amino acids. A mutation that results in the truncation of about two-thirds of the Gal4AD (gal4D) results in a crippled protein with only 3% the activity of the wild-type activator. We show here that although the Gal4D protein is not intrinsically deficient in DNA binding, it is nonetheless unable to stably occupy GAL promoters in vivo. This is because of the activity of the proteasomal ATPases, including Sug1/Rpt6, which bind to Gal4D via the remainder of the AD and strip it off of DNA. A mutation that suppressed the Gal4D "no growth on galactose" phenotype repressed the stripping activity of the ATPase complex but not other activities. We further demonstrate that Gal4D is hypersensitive to this stripping activity because of its failure to be monoubiquitylated efficiently in vivo and in vitro. Evidence is presented that the piece of the AD that is deleted in Gal4D protein is likely a recognition element for the E3 ubiquitin-protein ligase that modifies Gal4. These data argue that acidic ADs comprise at least two small peptide subdomains, one of which is responsible for activator monoubiquitylation and another that interacts with the proteasomal ATPases, coactivators and other transcription factors. This study validates the physiological importance of Gal4 monoubiquitylation and clarifies its major role as that of protecting the activator from being destabilized by the proteasomal ATPases.

Highlights

The proteasome is a large macromolecular complex that carries out most of the non-lysosomal proteolysis in eukaryotic cells [1]
The first hint of such a link was the finding that specific alleles of SUG1 and SUG2, genes that encode two of the proteasomal ATPases (Rpt4 and Rpt6), could rescue the activity of a Saccharomyces cerevisiae Gal4 transactivator derivative lacking about twothirds of the C-terminal activation domain (Gal4D) [5, 6]
Others later demonstrated that Sug1 and presumably other proteasomal ATPases are important in mediating histone extract; IMAC, immobilized metal affinity chromatography; CTR, C-terminal region; NTR, N-terminal region; qPCR, quantitative PCR; UAS, upstream activation sequence

Summary

EXPERIMENTAL PROCEDURES

Plasmids, and Proteins—Pre1-FLAG-tagged 26 S and 19 S have been described [19]. Yeast strains used for ChIP experiments were transformed with single-copy plasmids (derived from pSB32) expressing either wild-type Gal (pSJR263) or gal4D (pSJR261). Genetic fusion of ubiquitin to S10-tagged Gal4D in the pSB32 vector was done by removing Ub from GST-UbGap71-VP16 [17] using a NcoI digest and inserting Ub into the NcoI site at the start codon of the T7 tag For all of these constructs, the GAL4 gene was expressed from its own promoter. The sug S proteasome and congenic wild-type 26 S proteasome were purified as for the FLAG affinity tag except using T7-agarose (Novagen) and T7 peptide followed by a Mono-Q (Amersham Biosciences) column using a 20 mM–1 M NaCl gradient over 30 column volumes, which eluted proteasome at ϳ450 mM NaCl. GST-Gal4-(1–147)-VP16, GST-Gal4-(1–147), GST-Gal AD, GST-Gal4D AD, GST-ubiquitin-like domain of Rad23p, His6SUMO, His6-Gal, and His6-Ub were purified from Escherichia coli BL21 cells as described [8].

RESULTS

Identification of a Subdomain of the AD Required for

DISCUSSION