Maf1 is a conserved and central regulator of Pol III repression in all organisms tested, making its mode of regulation an issue of high interest. Here, the rapid repression of Pol III is likely important for conserving protein synthesis resources when unfavourable conditions are encountered. The rapid establishment of Pol III repression is correlated with the extent of Maf1 dephosphorylation in all conditions tested, and mutations that impair dephosphorylation impair repression, making the dephosphorylation mechanism a key feature of Maf1 regulation. Thus, it is critical to identify the correct phosphatase complex, the precise catalytic subunit, and which attendant subunits are needed for Maf1 dephosphorylation. Here, we establish PP4 as the major Maf1 phosphatase complex for acute Pol III repression, identify Pph3 as the involved catalytic subunit, identify the PP4 core and regulatory/interacting proteins involved in Maf1 dephosphorylation, show physical PP4 interaction with Maf1, and reconstitute PP4-dependent Maf1 dephosphorylation in vitro, providing evidence that the effect is direct ().
Our identification of PP4 as the major Maf1 phosphatase was enabled by a fusion protein, Maf1–Rpc160, which allowed us to test candidate PP2A-related complexes and components, and provided clear evidence for participation by certain PP4 members, but not PP2A. This was confirmed by extensive additional experiments on PP4 and PP2A, and their impact on Maf1 and Pol III repression in the natural (nonfused) context. Prior studies have shown that certain phosphorylation sites on Maf1 are involved in nuclear–cytoplasmic shuttling, raising the possibility that yeast simply use Maf1 phospho-dynamics to control the shuttling aspect of Maf1 regulation, but not the execution of Pol III repression in the nucleus. However, studies in yeast have shown that nuclear accumulation of Maf1 is insufficient to repress Pol III (Moir et al, 2006
; Towpik et al, 2008
). In addition, vertebrate Maf1, although under phosphoregulation, is constitutively nuclear (Kantidakis et al, 2010
). These results suggest that Maf1 dephosphorylation is critical for repression, but is not used to control nuclear localization in all species. We note that although overexpression of Maf1 confers modest Pol III repression, no growth defect is observed, suggesting that cells must cross a threshold of very low tRNA abundance before conferring a clear growth defect.
Our results add to our understanding of Maf1 localization during repression of Pol III. Although initial models suggested that Maf1 is dephosphorylated in the cytoplasm prior to nuclear localization, the mechanism is likely more complex. Maf1 contains two nuclear localization signals, one closer to the amino-terminus (NtNLS) and one closer to the carboxy-terminus (CtNLS). The NtNLS has been shown to be adjacent to serines phosphorylated by PKA and Sch9, and this NtNLS is rendered nonfunctional in the phosphorylated state (Moir et al, 2006
; Huber et al, 2009
; Lee et al, 2009
). However, based on studies of the msn5
Δ null, lacking the Maf1 exportin, Maf1 can be phosphorylated and dephosphorylated within the nucleus (Towpik et al, 2008
). Consistent with this, we find an essential subunit of PP4 complex, Psy2, located in the nucleus, supporting Maf1 dephosphorylation in the nucleus. Importantly, even in maf1
mutants with inactivated NtNLS (by either deletion of the NLS signal or phosphomimetic 6SE mutation), nuclear accumulation can occur readily upon stress (Moir et al, 2006
). In addition, since the CtNLS is not adjacent to known or predicted phosphorylation sites, it is possible that the CtNLS is constitutively active in Maf1 nuclear translocation, regardless of Maf1 phosphorylation state. One possible model involves constitutive, low-level translocation of phosphorylated Maf1 to the nucleus during favourable growth conditions, countered by constitutive export by Msn5. In unfavourable conditions, Maf1 is dephosphorylated in the nucleus, preventing its export by Msn5, thus leading to nuclear accumulation (). Importantly, in mutants where Maf1 is constitutively nuclear (e.g., msn5
), Pol III is not constitutively repressed, and maf1-7A
mutants respond robustly to rapamycin (Huber et al, 2009
), suggesting an additional required step beyond nuclear localization/retention, such as dephosphorylation at additional positions to confer Pol III repression. Although PP4 is clearly involved in nuclear localization of Maf1, it is also likely involved in this additional required step, since the fusion growth defect of maf1-7A–
Rpc160 is partially rescued in pph3
Δ mutants, and Pph3 is required for full Pol III repression by maf1-7A
mutant yeast in ND treatment (Supplementary Figure S8
Whereas multiple kinases act on Maf1, PP4 appears to mediate the majority of Maf1 dephosphorylation during the acute response to rapamycin, DNA damage, CPZ, CHX and ND—and is needed for the acute establishment of Pol III repression. (We note, however, that long-term Pol III repression appears to be independent of PP4, which likely involves the de novo translation and accumulation of unphosphorylated Maf1 over time.) Taken together, one straightforward interpretation of our data is that PP4 functions with Maf1 as a co-integrator of environmental conditions. To further test this, the interplay between the various Maf1 kinases and PP4 in favourable versus unfavourable growth conditions should be investigated. Specifically, it is not clear whether PP4 constitutively dephosphorylates Maf1—with the Maf1 kinases attenuated during stress—or whether PP4 activity towards Maf1 is increased in poor growth conditions. These models are not exclusive, and both could be involved. If PP4 is relatively constitutive, Maf1 activity could be kept in balance by kinase activation/deactivation in response to growth conditions, where poor growth conditions would lead to kinase inhibition, and PP4 removing the phosphorylated pool. Indeed, TORC1 activity is inhibited by ND and rapamycin, consistent with this model. However, in our view, some regulation of PP4 activity during stress seems likely, as a single type of stress (like DNA damage) causes Pol III repression even though the Maf1 kinases that assess nutrient availability remain active. Furthermore, there are known roles for TOR and Tap42 in regulating phosphatase activities. Our data does not support a major change in PP4–Maf1 interaction during stress, so if PP4 activation occurs, it likely involves improving its catalytic turnover. Therefore, understanding PP4 regulation and substrate selection is an important next step.
PP4 participates in the DNA damage response in multiple species, including yeast, human, Caenorhabditis elegans
(Cohen et al, 2005
; Gingras et al, 2005
; Kim et al, 2007
). This involves dephosphorylation of Rad53, which helps overcome G2/M arrest (O'Neill et al, 2007
). In Dictyostelium discoideum
, the PP4 complex is activated in response to starvation conditions to enable differentiation into fruiting bodies (Mendoza et al, 2005
). In addition to its role in the DNA damage response, C. elegans smk-1
(homologue of Psy2) is required for the response to oxidative and innate immune stress required for long-lived worms (Wolff et al, 2006
). Given the role for PP4 in the environmental stress response of higher organisms, a role for PP4 in Maf1 dephosphorylation in higher organisms should be explored.
Our findings also inform the compositional requirements of PP4 for dephosphorylation of Maf1. Here, PP4 ‘core' components Pph3 and Psy2 are required for dephosphorylation of Maf1, while Psy4 is not. Notably, Psy4 is required for dephosphorylation of γH2AX but not Rad53, while Psy2 is required for all known substrates (Keogh et al, 2006
; O'Neill et al, 2007
). Less is known regarding the use of ‘noncore' PP4 subunits in substrate selection, and here we identify Tip41 and Rrd1 as important for full Maf1 dephosphorylation. Tip41 acts in concert with PP4 in DNA damage (Gingras et al, 2005
) and activates Sit4 and PP2A catalytic subunits towards Gln3 (Jacinto et al, 2001
) and Msn2 (Santhanam et al, 2004
), respectively. Rrd1 and Rrd2 are yeast homologues of human phosphotyrosyl phosphatase activator (PTPA); Rrd1 activates several PP2A family phosphatases in vitro
, but selectively binds Pph3 (and not Pph21/22) in vivo
(Van Hoof et al, 2005
). PTPA has prolyl isomerase activity, which activates PP2A catalytic subunits (Jordens et al, 2006
). The proline residue involved in human PP2AC
is Pro190, which is conserved in Pph3, suggesting a conserved mechanism of activation.
Our data does not support a role for PP2A as the main phosphatase of Maf1 (Oficjalska-Pham et al, 2006
), or as a phosphatase needed for acute repression. In both tpd3
Δ (scaffold) and pph21
Δ (alternative catalytic) double mutants, Maf1 is completely dephosphorylated under stress conditions and with rapid kinetics, and the fusion growth defect is not rescued in these genetic backgrounds (; Supplementary Figure S4
). This is also true in rrd2
Δ mutants, which lack a PP2A-specific activator (). In fact, in PP2A mutants, we observe an enhanced fusion growth defect, suggesting that disruption of PP2A causes cellular stress, which elicits, rather than compromises, Pol III repression. The work can be reconciled by noting that all genotypes defective for Maf1 dephosphorylation involved combinations of pph21
along with pph3
Δ, which at the time was erroneously considered a redundant PP2A catalytic subunit; pph3
Δ was never examined in isolation, which we confirm here as exclusively a subunit of PP4. Although Pph3/PP4 is identified as the main and required Maf1 phosphatase, the slight Maf1 dephosphorylation observed during ND in pph3
Δ cells () leaves open the possibility for alternative minor phosphatases for Maf1 dephosphorylation.
Recently, the co-structure of Maf1 with yeast Pol III was solved (Vannini et al, 2010
), and showed Maf1 binding the clamp domain of Rpc160 (aa 1–245). Therefore, Maf1 fused to the amino-terminus of Rpc160 is an ideal position to poise the complex for repression. Binding of Maf1 causes a shift in the position of the C34 subunit that prevents Brf1 binding, effectively inhibiting TFIIIB–Pol III interactions. Other structural studies of Maf1 protein have shown that the phosphorylated form of Maf1 is correlated with absence of interaction between its amino-terminal A domain and its carboxyl-terminal BC domain, while dephosphorylation is correlated with their interaction (Gajda et al, 2010
). Thus, dephosphorylation may create a more compact Maf1 that binds in the pocket between C34 and C82 in the Pol III complex. These structural studies may help us understand the utility of our Maf1–Rpc160 fusion tool. First, the Maf1–Rpc160 fusion growth defect can be intensified to complete growth arrest by addition of 10 nM rapamycin (unpublished observations), or instead attenuated by deletion of PP4 phosphatase components. We suggest that in unfavourable growth conditions, Maf1 in the fusion context becomes fully dephosphorylated, enhancing its regulated interaction with the Pol III complex, further inhibiting Pol III interaction with TFIIIB and further decreasing transcription. Accordingly, in PP4 mutants, Maf1 is phosphorylated, eliminating A and BC domain interaction, allowing Brf1 association and normal Pol III activity and growth. We envision ourselves and others utilizing the Maf1–Rpc160 fusion to further investigate factors involved in Pol III repression.