The Gcd10p/Gcd14p complex is the essential two-subunit tRNA(1-methyladenosine) methyltransferase of Saccharomyces cerevisiae

Abstract

The modified nucleoside 1-methyladenosine (m(1)A) is found at position 58 in the TPsiC loop of many eukaryotic tRNAs. The absence of m(1)A from all tRNAs in Saccharomyces cerevisiae mutants lacking Gcd10p elicits severe defects in processing and stability of initiator methionine tRNA (tRNA(i)(Met)). Gcd10p is found in a complex with Gcd14p, which contains conserved motifs for binding S-adenosylmethionine (AdoMet). These facts, plus our demonstration that gcd14Delta cells lacked m(1)A, strongly suggested that Gcd10p/Gcd14p complex is the yeast tRNA(m(1)A)methyltransferase [(m(1)A)MTase]. Supporting this prediction, affinity-purified Gcd10p/Gcd14p complexes used AdoMet as a methyl donor to synthesize m(1)A in either total tRNA or purified tRNA(i)(Met) lacking only this modification. Kinetic analysis of the purified complex revealed K(M) values for AdoMet or tRNA(i)(Met) of 5.0 microM and 2.5 nM, respectively. Mutations in the predicted AdoMet-binding domain destroyed GCD14 function in vivo and (m(1)A)MTase activity in vitro. Purified Flag-tagged Gcd14p alone had no enzymatic activity and was severely impaired for tRNA-binding compared with the wild-type complex, suggesting that Gcd10p is required for tight binding of the tRNA substrate. Our results provide a demonstration of a two-component tRNA MTase and suggest that binding of AdoMet and tRNA substrates depends on different subunits of the complex.

Figure 1

The purified Gcd10p/Gcd14p complex has tRNA(m¹A)MTase activity specific for gcd10Δ tRNA. Affinity-purified FlagGcd10p/Gcd14p complexes were resolved on a Superose-6 FPLC column precalibrated with commercial standards of 4–670 kDa (Bio-Rad). The elution positions and masses of the standards are shown on top above the line. Aliquots containing equal portions of every other column fraction in the range predetermined to contain the complexes were separated by using SDS/PAGE followed by immunoblot analysis with antibodies against Gcd10p or Gcd14p (A) or by silver staining (B). Input lane contains an aliquot of the material loaded on the column. (C) Aliquots (3%) of the indicated fractions were assayed for (m¹A)MTase activity in reactions (0.05 ml) containing total tRNA (8.0 μM) isolated from yJA146 (gcd10Δ hcIMT4) or yJA158 (GCD10 hcIMT4) and S-adenosyl-l-[methyl-¹⁴C]methionine (10 μM). The acid-insoluble portion of each reaction was collected on nitrocellulose filters and dried, and the radioactivity was determined by liquid scintillation. The radioactivity incorporated is plotted against fraction number.

Figure 2

FlagGcd10p/Gcd14p complexes specifically catalyze synthesis of m¹A in purified tRNA_i^Met. Initiator tRNA^Met purified from yJA146 (gcd10Δ hcIMT4) was incubated with purified FlagGcd10p/Gcd14p complexes and [methyl-¹⁴C]AdoMet as described in Fig. 1, hydrolyzed to nucleosides, and resolved by HPLC chromatography as described (5). The fractions were monitored for UV absorbance at 254 nm (A₂₅₄; solid line) and assayed for cpm [¹⁴C-methyl] by flow scintillation analysis as described (31) (dotted line). (A) Mock-treated tRNA. (B) tRNA treated with FlagGcd10p/Gcd14p complexes. (C) Double reciprocal plots of tRNA(m¹A)MTase activity of purified FlagGcd10p/Gcd14p complexes as a function of tRNA_i^Met concentration (1.2–60 nM) (a) or Ado[¹⁴C]Met concentration (0.5–20 μM) (b). Assays contained 10 μM Ado[¹⁴C]Met (a) or 10 μM tRNA_i^Met (purified from the gcd10Δ hcIMT4 strain) (b) and approximately 0.3 μg of enzyme in each reaction (100 μl). Lines were fitted by using linear regression analysis.

Figure 3

Mutations in the AdoMet-binding motif I of Gcd14p eliminate m¹A synthesis in vivo. (A) Transformants of yeast strain F516 (gcd14–2) containing low-copy URA3 plasmids bearing wild-type GCD14 (p2599), GCD14Flag (pJA148), gcd14–3Flag (pJA149), or vector alone, were streaked on SC medium lacking uracil and incubated at 36°C for 2 days. (B) HPLC chromatographs of total tRNA isolated from yeast strain yJA203 (gcd14Δ hcIMT4) harboring pJA148 (GCD14Flag) or pJA149 (gcd14–3Flag). The A₂₅₄ was plotted against the time of elution. The identities of known nucleosides or their modified counterparts are indicated above or within the corresponding peaks (Ψ, pseudouridine; C, cytidine; U, uridine; m¹A; m⁵C, 5-methylcytidine).

Figure 4

In vitro tRNA(m¹A)MTase activity requires wild-type FlagGcd14p and Gcd10p. (A) FlagGcd14p/Gcd10p (lane 1), FlagGcd14–3p/Gcd10p (lane 2), or FlagGcd14p alone (lane 3) were affinity-purified from yeast cell extracts, separated by SDS/PAGE, and stained with Coomassie brilliant blue (Bio-Rad). Included on the gel are known amounts of BSA (lanes 4–6), and SeeBlue molecular weight standards (NOVEX). The positions of FlagGcd14p or Gcd10p are indicated. (B) Immunoblot analysis of increasing amounts of FlagGcd14p/Gcd10p (lanes 1–3), FlagGcd14–3p/Gcd10p (lanes 4–6), or FlagGcd14p alone (lanes 7–9) by using antibodies that recognize either FlagGcd14p or Gcd10p. (C) Samples of the purified proteins shown in A at the indicated concentrations were assayed for tRNA(m¹A)MTase activity by using Ado[methyl-³H]Met (30 μM) and tRNA_i^Met (150 nM) purified from yJA146 (gcd10Δ hcIMT4) as substrate. The acid-insoluble cpm produced in each reaction was determined at the indicated times, converted into pmol of [methyl-³H]tRNA_i^Met, and plotted against time.

Figure 5

Evidence that tRNA binding by Gcd14p/Gcd10p requires Gcd10p. The indicated purified FlagGcd14p/Gcd10p complexes (A and B) or FlagGcd14p alone (C) or comparable amounts of protein purified from untagged (GCD14, D) were combined in TB binding buffer with [³²P]tRNA_i^Met purified from the gcd10Δ hcIMT4 strain. Reactions were filtered on a slot blot apparatus containing a three-membrane sandwich, as described in Materials and Methods. The cpm of ³²P retained on the second membrane (nitrocellulose), which typically contains specific RNA-protein complexes, was quantified by phosphorimaging analysis using a Storm 860 apparatus and imagequant software. The graph shows the relative amount of bound [³²P]tRNA_i^Met plotted against the protein concentration for each reaction, with columns 1–5 designating reactions containing protein concentrations of 0, 8, 16, 32, or 64 nM, respectively. (Inset) An autoradiogram of the nitrocellulose membrane used to generate the graphical data. The rows A–D and columns 1–5 designate the identities of the complex and protein concentrations, respectively, as described in the graph.