Investigation of the impact of Maf1 on yeast physiology indicates that Maf1 performs a significant inhibitory role in normally growing cells. This new function of Maf1 couples Pol III transcription with metabolic processes and/or energy production that is dependent on the carbon source. It is now obvious that not only the Pol II transcriptome but also the Pol III genes are regulated during the transition from fermentative to glycerol-based respiratory growth and that Maf1 is essential for this regulation. Previous work indicates that dephosphorylation is the way by which nuclear importation and Maf1 activity are regulated in response to rapamycin. Here we show that Maf1 is phosphorylated and localized in a manner that is dependent on the carbon source, which determines yeast metabolism. Glucose depletion and transfer to a nonfermentable carbon source result in Maf1 dephosphorylation and importation into the nucleus. An opposite transition, from a nonfermentable carbon source to a glucose medium, is followed by Maf1 phosphorylation and relocation from the nucleus to the cytoplasm. It is not clear if the phosphorylation occurs before or after Maf1 export to the cytoplasm. Our kinetics studies suggest that relocalization to the cytoplasm is coincident with Maf1 phosphorylation. The mechanism of Maf1 nuclear exportation remains to be determined.
Maf1 of S. cerevisiae
is a serine-rich protein (15.7% of serines) and, according to a Swiss-Prot PROSITE search, contains 30 potential phosphorylation sites. The kinase involved in Maf1 phosphorylation following a transfer to glucose is currently unknown. The cAMP-dependent PKA kinase was a potential candidate since Maf1 had already been experimentally confirmed as its substrate in vitro (3
). We therefore tested strains carrying the mutations RAS2Val19
, each of which affected the synthesis of cAMP differently. We found that neither mutation altered the pattern of Maf1 phosphorylation following transfer to a different carbon source. No effect of cdc25-1
on the kinetics of Maf1 phosphorylation upon transfer to glucose medium was observed (data not shown). Moreover, the high PKA activity in the RAS2Val19
mutant did not limit the dephosphorylation and nuclear relocation of Maf1 in response to glucose depletion. In conclusion, we suggest that another, so-far-unknown kinase participates in the regulation of Maf1 activity toward the coupling of Pol III transcription with carbon metabolism.
Having established that an altered level of PKA activity did not affect Maf1 phosphorylation or localization upon transition from glucose to a nonfermentable carbon source, we also expected no effect on the regulation of Pol III activity during such a transition. However, a direct study of pre-tRNA synthesis in the PKA mutants resulted in an opposite conclusion. The high PKA activity in the RAS2Val19
cells grown in glucose medium and transferred to respiratory conditions prevented a decrease in Pol III transcription. Consistently, in the cdc25-1
mutant with a low PKA activity, Pol III activity was more repressed than in the corresponding wild type. A similar effect of altered PKA activity in mutants was previously observed by Moir et al. (23
) on the repression of Pol III transcription upon rapamycin stress, but the authors reported an accompanying change in the pattern of Maf1 phosphorylation. Here, we clearly show that the same mutants which do not affect the phosphorylation and cellular localization of Maf1 do affect, under the same conditions, the regulation of Pol III activity. As suggested previously (23
), there is another protein involved in Pol III transcription regulation. According to our hypothesis, this unknown protein might be directly regulated by PKA.
Our current results show that not all Pol III genes are regulated by Maf1 to the same extent when cells are grown in a nonfermentable carbon source. This conclusion is confirmed by quantification of a Pol III-specific microarray and by quantitative reverse transcription-PCR with selected tRNA genes. The mechanism and physiological basis of the variable Maf1 regulation of individual tRNAs remain unclear. Assuming a direct influence of Maf1 on the transcription rate, the variable effect of Maf1 on different tRNA genes could be dependent on the potential transcription efficiency of a given tRNA gene. tRNA genes with internal Pol III promoters are homologous, but the flanking sequences are different. This might be the reason for different occupancy of TFIIIB and Pol III (13
). It has also been shown that the RSC chromatin remodeling complex is specific to Pol III genes, but not all tRNA genes bind RSC (24
). The relative efficiency of transcription of individual tRNA genes is probably not the same, although this problem has not yet been solved. Therefore, we were not able to establish the relationship between the effect of Maf1 and the transcription efficiency of a particular tRNA gene.
The extent of Maf1 regulation does not correlate with the gene copy number for a given tRNA (see Table S1 in the supplemental material). The expression of single-copy tRNA genes is usually not much affected in maf1-Δ cells. However, the levels of tRNAVal (tV[AAC]E1, 13 copies) or tRNAAsn (tN[GUU]C, 10 copies) were also affected to a minor extent, whereas those of tRNAPhe (tF[GAA]N, 2 copies) and tRNAArg (tR[ACG]J, 1 copy) were significantly increased in maf1-Δ cells grown under respiratory conditions. We also found no correlation between the extent of Maf1 regulation and codon usage corresponding to a given tRNA.
Unbalanced tRNA levels seem a likely reason for the maf1
-Δ growth defect in glycerol medium. In contrast to some other tRNAs, initiator tRNAMet
was not significantly affected by Maf1 since its expression ratio in a nonfermentable carbon source was 2.65 ± 1.23. It is known that depletion of initiator tRNAMet
triggers reprogramming of genome expression in several Pol III mutants (5
). Assuming that the relatively low level of initiator tRNAMet
could be the reason for growth inhibition, its expression was increased by introducing a multicopy plasmid with the IMT1
gene into the cells. However, no effect of the increased dose of initiator tRNAMet
on the maf1
-Δ phenotype was observed (data not shown). In S. cerevisiae
, two Pol III-synthesized tRNAs have been reported as mitochondrially targeted, namely tRNALys
). The mitochondrial functions of these tRNAs are not fully clear, although there is indirect evidence for their role in mitochondrial translation. Our data show that tRNALys
is 6.03- ± 2.44-fold increased in maf1
-Δ, whereas tRNAGln
is not affected. Assuming that the imported tRNAs function in mitochondria in a concerted fashion, their unbalanced levels could be disadvantageous for mitochondrial translation. However, no increased [rho−
] accumulation, typical for yeast mutants with mitochondrial translation defects, was observed in maf1
Although nuclear Maf1 is known to function as a Pol III transcription repressor, the role of Maf1 in the cytoplasm remains unknown. One possibility, involving the mitochondrial scenario, is a function of cytoplasmic Maf1 in posttranscriptional tRNA control. At least two of the four subunits of the yeast tRNA endonuclease, Sen2 and Sen54, are located on the outer mitochondrial membrane, and this location is important for functional tRNA splicing (44
). Although no mitochondrial phenotype of mutants affecting tRNA splicing has been found, one could assume that cytoplasmic Maf1 could somehow be involved. Interestingly, we identified the Sen54-encoding gene in a screen for putative activators of tRNA biosynthesis (M. Cieśla, unpublished). Nonetheless, inactivation of MAF1
is not the first example of a mutation that alters tRNA biogenesis and the ability of yeast cells to grow in respiratory substrates. A partial deletion of the N-terminal domain of τ55, a TFIIIC subunit required for tRNA transcription, impairs its function and also cell growth at an elevated temperature in nonfermentable carbon sources (20
The lethality of the maf1
-Δ cells under restrictive conditions is caused by increased or unbalanced levels of some Pol III products because it can be overcome by decreased transcription in Pol III mutants. The maf1
-Δ growth defect could be suppressed by selected mutations affecting Pol III transcription initiation or termination. Suppression of the temperature-sensitive phenotype of maf1
-Δ in Pol III mutants may reflect simple compensation of the amount of active transcription complexes. Since not all Pol III mutants with similar decreases (n
-fold) in tRNA levels suppressed maf1
-Δ, we propose that only some mutations allow formation of functional Pol III complexes in the absence of Maf1. Interestingly, we found a reciprocal genetic interaction of Maf1 with the C31 Pol III subunit. Truncation of the C31 subunit in the rpc31-236
mutant caused a temperature-sensitive phenotype (38
counteracted the maf1
-Δ defect in a nonfermentable carbon source. Interestingly, maf1
was no longer thermosensitive, indicating that in the absence of a negative regulator, the C31 truncation was not detrimental to Pol III transcription activity at an elevated temperature. C31 is part of a subcomplex of three Pol III-specific subunits (C31, C34, and C82) that is thought to interact with TFIIIB (41
). The genetic interaction of Maf1 and C31 supports the model in which Maf1 affects the recruitment of Pol III by hampering its interaction with TFIIIB. Moreover, the gene encoding the Ded1 helicase was previously found to be a suppressor of rpc31-236
). Ded1 is another putative link since it was immunopurified with Maf1 (25
). These preliminary genetic data encourage us to study the mechanism of action of Maf1 on the Pol III transcription apparatus.