3.1.2. Ribosomal Protein Production: Ifh1, Crf1 and Spf1
The second major regulon in ribosome production are transcripts coding for ribosomal proteins (RP). As for RiBi genes, rapamycin treatment causes severe repression of this regulon
[21,99][21][99]. The main transcription factors involved in RP gene transcription are Rap1, Hmo1
[100], Sfp1 and Fhl1 with its co-factors Ifh1 and Crf1
[101,102,103][101][102][103]. Of these, Rap1, Sfp1 and Fhl1-Ifh1/Crf1 have been linked to the TOR and PKA pathways
[101,102,104,105,106][101][102][104][105][106]. Additionally, Stb3, functions as a transcriptional repressor also of RP genes, albeit apparently not via the binding of RRPE promoter motifs
[24,88][24][88] (
Figure 2). PKA-dependent regulation of RP genes induced by Rap1 has been shown in a number of experiments
[106,107][106][107]: RP-transcripts were up-regulated in a Rap1-dependent manner in a
BCY1 deletion strain and the presence of the Rap1 binding site in its promoter was sufficient to confer an increase in a reporter transcript in a
bcy1∆ strain compared to wt
[108]. While phosphorylation of Rap1 via PKA cannot be completely ruled out, more recent models suggest, Rap1 serves as a recruiting factor for proteins regulated by PKA. These binding partners may include Sfp1 and Fhl1 and its co-factors
[107].
While 127 of the 138 RP genes harbor Rap1-binding sites, approximately a half are bound by Fhl1
[101,107][101][107]. Fhl1 is not apparently regulated by PKA or TOR, instead, regulation occurs on the level of its co-regulators Ifh1 and Crf1. The level of the Ifh1 bound to RP genes drops upon nutrient depletion or rapamycin treatment and increases when cells resume growth upon encountering improved nutrient conditions, while Fhl1 remains constitutively associated with the promoters
[101,102,103][101][102][103]. The interaction of Fhl1 and Ifh1 depends on the forkhead-associated (FHA) domain of Fhl1
[101[101][102],
102], a domain previously reported to interact with phosphorylated sequences
[109]. It is, therefore, reasonable to posit that Fhl1-Ifh1 interaction depends on Ifh1 phosphorylation. Martin, 2004 provided a detailed description of how transcriptional activator Ifh1 and repressor Crf1 compete for binding to Fhl1
[104]. They observed a rapamycin-triggered increase in Crf1 phosphorylation, causing its nuclear translocation and therefore RP gene repression. As this process could be prevented by mutations that render PKA signaling hyperactive, they suggested that TORC1 acts upstream of PKA in this respect
[104]. They further found that protein kinase Yak1 is required for Crf1 nuclear translocation. As Yak1 is a known direct PKA, but not TORC1/Sch9 target, this is also consistent with the idea of TORC1 regulating PKA.
Surprisingly, regulation of RP gene transcription by Crf1 appears to be yeast strain dependent, as Zhao et al. observed no effect of
CRF1 deletion on RP transcription in the W303, while they did observe the effect in the TB50 strain
[110]. The Yak1-dependent mechanism also appears to be at odds with the finding by others that RP gene transcription is still repressed by rapamycin in strains lacking Yak1
[72].
IResearcher propose that this is explained by a secondary mechanism that acts on longer timescales. Indeed, remaining RP gene inhibition in strains lacking
CRF1 was observed also in the original study by Martin, 2004. What this second mechanism may be is not yet clear. Finally, to date, only CK2 has been shown to phosphorylate Ifh1 and Crf1 in a manner relevant for Fhl1 binding
[111,112][111][112]. Mechanistic details if and how PKA and TORC1 regulate Fhl1-dependent RP transcription are therefore still lacking.
Sfp1 is a transcription activator of RP genes and deletion strains of SFP1 are marked by a striking small cell size phenotype
[113]. While Sfp1 binds promoters of other gene groups as well, this appears to be mechanistically different
[114].
Sfp1 was associated with TOR signaling in a microscopic screen as it localized from the nucleus to the cytoplasm upon rapamycin treatment
[115]. Importantly, PKA signaling can override TORC1 effects to some extent as hyperactivated PKA (
bcy1∆) largely counteracted the rapamycin-induced cytoplasmic relocalization. In which way do PKA and TOR signaling converge on Sfp1? Sfp1 phosphorylation was reduced upon rapamycin treatment and, in vitro phosphorylation by TORC1 was observed, while no phosphorylation was detected in an in vitro kinase assay with Sch9
[105]. This renders Sfp1 one of only a handful of direct TORC1 substrates. Seven potential TORC1 phosphorylation sites on Sfp1 were explored and their mutation almost completely abrogated further dephosphorylation upon rapamycin treatment
[105]. Sfp1 was also phosphorylated by bovine PKA in vitro, but phosphorylation sites were not determined
[97]. An RRxS motif at a site different from the TORC1-dependent sites and conserved in multiple yeast species was proposed to constitute an important PKA site
[97]. Regulation of Sfp1 through common sites by PKA and TORC1 is therefore unlikely.