Joanna Towpik

Maf1 Is Involved in Coupling Carbon Metabolism to RNA Polymerase III Transcription

ABSTRACT RNA polymerase III (Pol III) produces essential components of the biosynthetic machinery, and therefore its activity is tightly coupled with cell growth and metabolism. In the yeast Saccharomyces cerevisiae, Maf1 is the only known global and direct Pol III transcription repressor which mediates numerous stress signals. Here we demonstrate that transcription regulation by Maf1 is not limited to stress but is important for the switch between fermentation and respiration. Under respiratory conditions, Maf1 is activated by dephosphorylation and imported into the nucleus. The transition from a nonfermentable carbon source to that of glucose induces Maf1 phosphorylation and its relocation to the cytoplasm. The absence of Maf1-mediated control of tRNA synthesis impairs cell viability in nonfermentable carbon sources. The respiratory phenotype of maf1-Δ allowed genetic suppression studies to dissect the mechanism of Maf1 action on the Pol III transcription apparatus. Moreover, in cells grown in a nonfermentable carbon source, Maf1 regulates the levels of different tRNAs to various extents. The differences in regulation may contribute to the physiological role of Maf1.

Derepression of RNA polymerase III transcription by phosphorylation and nuclear export of its negative regulator, Maf1.

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-Δ 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-Δ 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.

Maf1 Protein, Repressor of RNA Polymerase III, Indirectly Affects tRNA Processing

Maf1 is negative regulator of RNA polymerase III in yeast. We observed high levels of both primary transcript and end-matured, intron-containing pre-tRNAs in the maf1Δ strain. This pre-tRNA accumulation could be overcome by transcription inhibition, arguing against a direct role of Maf1 in tRNA maturation and suggesting saturation of processing machinery by the increased amounts of primary transcripts. Saturation of the tRNA exportin, Los1, is one reason why end-matured intron-containing pre-tRNAs accumulate in maf1Δ cells. However, it is likely possible that other components of the processing pathway are also limiting when tRNA transcription is increased. According to our model, Maf1-mediated transcription control and nuclear export by Los1 are two major stages of tRNA biosynthesis that are regulated by environmental conditions in a coordinated manner.

Full Repression of RNA Polymerase III Transcription Requires Interaction between Two Domains of Its Negative Regulator Maf1

Maf1, first identified in yeast Saccharomyces cerevisiae, is a general negative regulator of RNA polymerase III (Pol III). Transcription regulation by Maf1 is important under stress conditions and during the switch between fermentation and respiration. Maf1 is composed of two domains conserved during evolution. We report here that these two domains of human Maf1 are resistant to mild proteolysis and interact together as shown by pull-down and size-exclusion chromatography and that the comparable domains of yeast Maf1 interact in a two-hybrid assay. Additionally, in yeast, a mutation in the N-terminal domain is compensated by mutations in the C-terminal domain. Integrity of both domains and their direct interaction are necessary for Maf1 dephosphorylation and subsequent inhibition of Pol III transcription on a nonfermentable carbon source. These data relate Pol III transcription inhibition to Maf1 structural changes.

Yeast prion [PSI+] lowers the levels of mitochondrial prohibitins

We report proteomic analyses that establish the effect of cytoplasmic prion [PSI(+)] on the protein complement of yeast mitochondria. A set of 44 yeast mitochondrial proteins whose levels were affected by [PSI(+)] was identified by two methods of gel-free and label-free differential proteomics. From this set we focused on prohibitins, Phb1 and Phb2, and the mitochondrially synthesized Cox2 subunit of cytochrome oxidase. By immunoblotting we confirmed the decreased level of Cox2 and reduced mitochondrial localization of the prohibitins in [PSI(+)] cells, which both became partially restored by [PSI(+)] curing. The presence of the [PSI(+)] prion also caused premature fragmentation of mitochondria, a phenomenon linked to prohibitin depletion in mammalian cells. By fractionation of cellular extracts we demonstrated a [PSI(+)]-dependent increase of the proportion of prohibitins in the high molecular weight fraction of aggregated proteins. We propose that the presence of the yeast prion causes newly synthesized prohibitins to aggregate in the cytosol, and therefore reduces their levels in mitochondria, which in turn reduces the stability of Cox2 and possibly of other proteins, not investigated here in detail.

Mitochondrial release factor in yeast: interplay of functional domains

The MRF1 gene encodes the only class I release factor found in Saccharomyces cerevisiae mitochondria, mRF1. The previously isolated point mutation mrf1-13 caused respiratory deficiency due to inhibition of mitochondrial translation. In this study, we have isolated second-site suppressors of mrf1-13. Among over 200 respiratory positive suppressor colonies, ten nuclear dominant suppressors had a new mutation in the MRF1 gene. The suppressors in combination with the original mrf1-13 revealed increased levels of mitochondrially synthesized proteins, Cox2 and Atp6. One of the suppressor alleles was cloned on a plasmid and was found to support weaker respiratory competence than in combination with mrf1-13. Finally, the possible effects of the suppressor mutations are discussed based on a structural model of mRF1 protein built for its “open” and “closed” forms using known crystal structures of prokaryotic release factor RF1 as templates. The 3D models suggest that at least some suppressors switch the structure of mRF1 from the “closed” to a permanently “open” form causing stronger binding of the mRF1 protein to the ribosome and increasing the time of ribosome occupation. This explains how the suppressor mutants may facilitate translation termination despite a defect in decoding of the stop signal.

Predicting proteome dynamics using gene expression data

While protein concentrations are physiologically most relevant, measuring them globally is challenging. mRNA levels are easier to measure genome-wide and hence are typically used to infer the corresponding protein abundances. The steady-state condition (assumption that protein levels remain constant) has typically been used to calculate protein concentrations, as it is mathematically convenient, even though it is often not satisfied. Here, we propose a method to estimate genome-wide protein abundances without this assumption. Instead, we assume that the system returns to its baseline at the end of the experiment, which is true for cyclic phenomena (e.g. cell cycle) and many time-course experiments. Our approach only requires availability of gene expression and protein half-life data. As proof-of-concept, we predicted proteome dynamics associated with the budding yeast cell cycle, the results are available for browsing online at http://dynprot.cent.uw.edu.pl/. The approach was validated experimentally by verifying that the predicted protein concentration changes were consistent with measurements for all proteins tested. Additionally, if proteomic data are available as well, we can also infer changes in protein half-lives in response to posttranslational regulation, as we did for Clb2, a post-translationally regulated protein. The predicted changes in Clb2 abundance are consistent with earlier observations.