Elongator function depends on antagonistic regulation by casein kinase Hrr25 and protein phosphatase Sit4

Abstract

SummaryIn yeast, the role for the Elongator complex in tRNA anticodon modification is affected by phosphorylation of Elongator subunit Elp1. Thus, hyperphosphorylation of Elp1 due to inactivation of protein phosphatase Sit4 correlates with Elongator-minus phenotypes including resistance towards zymocin, a tRNase cleaving anticodons of Elongator-dependent tRNAs. Here we show that zymocin resistance of casein kinase hrr25 mutants associates with hypophosphorylation of Elp1 and that nonsense suppression by the Elongator-dependent SUP4 tRNA is abolished in hrr25 or sit4 mutants. Thus changes that perturb the evenly balanced ratio between hyper-and hypophosphorylated Elp1 forms present in wild-type cells lead to Elongator inactivation. Antagonistic roles for Hrr25 and Sit4 in Elongator function are further supported by our data that Sit4 inactivation is capable of restoring both zymocin sensitivity and normal ratios between the two Elp1 forms in hrr25 mutants. Hrr25 binds to Elongator in a fashion dependent on Elongator partner Kti12. Like sit4 mutants, overexpression of Kti12 triggers Elp1 hyperphosphorylation. Intriguingly, this effect of Kti12 is blocked by hrr25 mutations, which also show enhanced binding of Kti12 to Elongator. Collectively, our data suggest that rather than directly targeting Elp1, the Hrr25 kinase indirectly affects Elp1 phosphorylation states through control of Sit4-dependent dephosphorylation of Elp1.

Use of a Yeast TRNase Killer Toxin to Diagnose Kti12 Motifs Required for TRNA Modification by Elongator

“…These observations again support our previously presented working hypothesis, that direct physical contact with Kti12 is required for Elongator's ability to modify tRNA anticodons [6,28,29]. …”

“…To avoid abundance related secondary effects we conducted subsequent analyses with the latter two mutants. Since Kti12 and Elongator associate with each other [6,24,28,36], we examined whether inappropriate interaction between both may underlie the loss-of-function traits observed in kti12-2 and kti12-8 cells (Figure 1 and 2). Therefore, c-Myc-tagged KTI12, kti12-2 and kti12-8 alleles ( Figure 3A) were each co-expressed with an HA-tagged copy of the ELP2 gene for co-immune precipitation (IP) assays ( Figure 3B).…”

“…Use of the -toxin tRNase as a tool diagnostic for Elongator activity identified additional class II gene products with related roles in tRNA modification [2,5,19,[23][24][25]. Among them, a casein kinase I isozyme (Hrr25/Kti14), a protein phosphatase (Sit4) and an Elongator partner protein (Kti12) were found to constitute a complicated network that regulates the phosphorylation status of Elongator's largest subunit (Elp1) [26][27][28]. This suggested that phosphorylation affects Elongator activity, a notion supported by data showing that Hrr25 phosphorylation sites on Elp1 are critical for U34 modification [29] and that Hrr25 association with Elongator requires Kti12 [28].…”

Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes

“…60 The kinase Kti14p and the phosphatase Sit4p seem to antagonistically regulate activity of Elongator complex by phosphorylation/ de-phosphorylation of the largest Elongator subunit Elp1p. 60,61 In addition to phosphorylation/ de-phosphorylation, the activity of Elongator complex also seems to be regulated by proteolysis of Elp1p. 62 The Kti11p interacts with Elongator complex through its C-termini and loss of Kti11p enhances the proteolysis of Elp1p.…”

A fungal anticodon nuclease ribotoxin exploits a secondary cleavage site to evade tRNA repair

“…The wobble modification is required for tRNA cleavage by g-toxin; yeast mutants that lack any of the enzymes responsible for synthesizing the mcm 5 moiety are resistant to zymocin (Frohloff et al 2001;Huang et al 2005;Lu et al 2005;Jablonowski et al 2006;Huang et al 2008). The latter includes the Elongator subunits (Elp1-6) and Elongator interacting proteins Kti11, Kti12, and Kti13, as well as the methylase complex Trm9-Trm112 and the phosphatase Sit4 and kinase Kti14 (Jablonowski et al 2004;Jablonowski and Schaffrath 2007;Huang et al 2008;Studte et al 2008;Mehlgarten et al 2009). Individual loss of the wobble uridine 2-thio modification of tRNA Glu provides only partial zymocin resistance and weak cleavage stimulation in vitro (Huang et al 2008;Lu et al 2005), indicating the mcm 5 side chain, but not the 2-thio group of mcm 5 s 2 U, to be crucial for tRNA cleavage by g-toxin.…”