Use of a Yeast tRNase Killer Toxin to Diagnose Kti12 Motifs Required for tRNA Modification by Elongator

Abstract

Saccharomyces cerevisiae cells are killed by zymocin, a tRNase ribotoxin complex from Kluyveromyces lactis, which cleaves anticodons and inhibits protein synthesis. Zymocin's action requires specific chemical modification of uridine bases in the anticodon wobble position (U34) by the Elongator complex (Elp1-Elp6). Hence, loss of anticodon modification in mutants lacking Elongator or related KTI (K. lactis Toxin Insensitive) genes protects against tRNA cleavage and confers resistance to the toxin. Here, we show that zymocin can be used as a tool to genetically analyse KTI12, a gene previously shown to code for an Elongator partner protein. From a kti12 mutant pool of zymocin survivors, we identify motifs in Kti12 that are functionally directly coupled to Elongator activity. In addition, shared requirement of U34 modifications for nonsense and missense tRNA suppression (SUP4; SOE1) strongly suggests that Kti12 and Elongator cooperate to assure proper tRNA functioning. We show that the Kti12 motifs are conserved in plant ortholog DRL1/ELO4 from Arabidopsis thaliana and seem to be involved in binding of cofactors (e.g., nucleotides, calmodulin). Elongator interaction defects triggered by mutations in these motifs correlate with phenotypes typical for loss of U34 modification. Thus, tRNA modification by Elongator appears to require physical contact with Kti12, and our preliminary data suggest that metabolic signals may affect proper communication between them.

Keywords: Elongator complex; Kti12; ribotoxin; tRNA anticodon modification; tRNase; zymocin.

Figure 1

A set of kti12 mutants uncovers a phenotypic signature typical of Elongator defects. (A) compilation of kti12 mutants including mutation mapping and alignment to Kti12 domain organization (for details, see Figure S1); (B,C) phenotypes shared between kti12 and Elongator mutants. Growth assays (B) with yeast strains of the indicated backgrounds at 30 °C (control), elevated temperature (38 °C) or in the presence of 45% [v/v] zymocin (+zymo). Traits typical of Elongator mutant (elp3∆) include zymocin resistance (R) and thermosensitivity (+); zymocin sensitivity (S) and thermotolerance (−) denote wild-type (KTI12) phenotypes; (C) phase contrast microscopy to monitor cell/bud morphology in the indicated strain backgrounds.

Figure 2

KTI12 gene mutations and copy number interfere with U34 tRNA suppressors. (A) tRNA nonsense suppression. In the indicated genetic backgrounds, can1-100 and ade2-1 ochre read-through by SUP4 was phenotypically assessed by canavanine sensitivity/resistance (S/R: middle panel) on canavanine supplemented medium lacking arginine (+can −arg) or by adenine proto-/auxotrophy (+/−: bottom panel) on minimal medium without adenine (−ade). Growth control (top panel) involved yeast peptone dextrose (YPD) rich medium. Empty vector (ev) served as reference for multi-copy (mc) KTI12; (B) SOE1 tRNA missense suppression. The indicated strains were grown at temperatures permissive (30 °C) or restrictive (36 °C) for cdc8-1^ts cells. Note that ELP1 and KTI12 gene mutations affect (+) rescue of cdc8-1^ts cell growth at 36 °C (−) by the tRNA suppressor SOE1.

Figure 3

Kti12 expression and Elongator interaction studies. (A) expression of Kti12 and its mutated variants. The indicated KTI12/kti12 alleles were genomically c-Myc-tagged and each gene product identified (top panel) in anti-c-Myc Western blots of total yeast protein extracts. An independent, anti-Cdc19 blot served as internal control (bottom panel); (B) elongator interaction studies with Kti12 and its mutants. Strains co-expressing Elp2-HA and c-Myc-tagged Kti12, Kti12-2 or Kti12-8 were subjected to anti-HA immune precipitation (IP). The IPs were probed for Elp2-HA (top panel) or Kti12-c-Myc (middle panel) with anti-HA or anti-c-Myc antibodies, respectively. Total protein extracts (preIP, bottom panel) probed with anti c-Myc antibodies served as Kti12 input control.

Figure 4

CaM binding by Kti12 in vitro. (A) CaM affinity chromatography of Kti12 and Kti13 variants from the indicated genetic backgrounds. Equal proportions of input (load), unbound (flow-through; washes) and eluted (EGTA elution) protein fractions were subjected to anti-TAP (top panel) and anti c-Myc Western blots (other panels); (B) killer eclipse assays between the same S. cerevisiae strains used for CaM affinity chromatography and a K. lactis zymocin producer (killer strain). ‘S’ or ‘R’ denote zymocin sensitivity or resistance.

Figure 5

KTI12 and ELO4 gene shuffle analysis. (A) A PSTK homolog-based structural model of yeast Kti12 with highlighted conserved P-loop (blue) and CBD1 (pink) in cartoon representation. PSTK based structures of P-loop (PL) and CBD1 from ELO4 provide a structural insight into the motif swap experiment. Highly conserved residues are shown in ball and stick representation, and labelled respectively; (B) Indication of PL and CBD motif shuffling from ELO4 to Kti12; (C,D) cross-complementation was studied by (C) conditional γ-toxin tRNase expression [6] on glucose (γ-toxin: off) vs. galactose (γ-tox: on) and (D) SUP4 nonsense suppression of ade2-1 (see also Figure 2A). R/S: γ-toxin resistance/sensitivity; +/−: adenine proto-/auxotrophy; (E) elongator interaction. Strains co-expressing Elp1-HA and indicated c-Myc-tagged Kti12 variants were subjected to anti-HA immune precipitation (IP). The IPs were probed with anti-HA and anti-c-Myc antibodies to check for content of Elp1-HA (top panel) and co-precipitated Kti12 material (middle panel). Total extracts (bottom panel) were probed with anti-c-Myc antibodies and served as input (preIP) control.