Derepression of RNA Polymerase III Transcription by Phosphorylation and Nuclear Export of Its Negative Regulator, Maf1

Joanna Towpik,Damian Graczyk,Anna Gajda,Olivier Lefebvre,Magdalena Boguta

doi:10.1074/jbc.m709157200

Abstract

Maf1 is the global repressor of RNA polymerase III (Pol III) in yeast Saccharomyces cerevisiae. Transcription regulation by Maf1 is important under stress conditions and during the switch between fermentation and respiration. Under repressive conditions on nonfermentable carbon sources, Maf1 is dephosphorylated and located predominantly in the nucleus. When cells were shifted to glucose medium, Maf1 became phosphorylated and concomitantly relocated to the cytoplasm. This relocation was dependent on Msn5, a carrier responsible for export of several other phosphoproteins out of the nucleus. Using coimmunoprecipitation, Maf1 was found to interact with Msn5. When msn5-Delta cells were transferred to glucose, Maf1 remained in the nucleus. Remarkably, despite constitutive presence in the nucleus, Maf1 was dephosphorylated and phosphorylated normally in the msn5-Delta mutant, and Pol III was under proper regulation. That phosphorylation of Maf1 and Pol III derepression are tightly linked was shown by studying tRNA transcription in Maf1 mutants with an altered pattern of phosphorylation. In summary, we conclude that phosphorylation of Maf1 inside the nucleus acts both directly by decreasing of Maf1-mediated repression of Pol III and indirectly by stimulation of Msn5 binding and export of nuclear Maf1 to the cytoplasm.

Highlights

We show that not Maf1 nuclear export but, rather, Maf1 phosphorylation is necessary for derepression of polymerase III (Pol III) transcription
Relocation of Maf1 to the cytoplasm in response to glucose appeared to be concomitant with Maf1 hyperphosphorylation, suggesting a link between the Maf1 phosphorylation state and its cellular location
RNA Pol III produces essential components of the protein biosynthesis machinery, and its activity is tightly coupled to cell growth and metabolism

Summary

EXPERIMENTAL PROCEDURES

Plasmids, and Medium—Yeast strains used in this study included BY4741 Cells were broken with acid-washed glass beads, the supernatant was retained, and trichloroacetic acid-precipitated proteins were pelleted by centrifugation. The membrane was blocked for 30 min in 10 mM Tris, 150 mM NaCl, and 0.05% Tween 20 containing 5% fat-free dry milk and incubated for 1 h with Maf1-specific antibody at a 1:10,000 dilution (8). Coimmunoprecipitation—Yeast cells expressing wild-type or HA epitope-tagged versions of Msn were grown in YPD to exponential phase, transferred to prewarmed YPGly for 1 h, transferred into YPD, incubated for 30 min, and pelleted and frozen in liquid nitrogen. After extensive washing in phosphate-buffered saline containing 0.5% bovine serum albumin and in buffer H, the beads were incubated with 2 mg of protein extract for 4 h with gentle shaking at 4 °C.

RESULTS

Derepression of Pol III Is Correlated with Phosphorylated State of

DISCUSSION