Saccharomyces cerevisiae possesses a stress-inducible glycyl-tRNA synthetase gene

Abstract

Aminoacyl-tRNA synthetases are a large family of housekeeping enzymes that are pivotal in protein translation and other vital cellular processes. Saccharomyces cerevisiae possesses two distinct nuclear glycyl-tRNA synthetase (GlyRS) genes, GRS1 and GRS2. GRS1 encodes both cytoplasmic and mitochondrial activities, while GRS2 is essentially silent and dispensable under normal conditions. We herein present evidence that expression of GRS2 was drastically induced upon heat shock, ethanol or hydrogen peroxide addition, and high pH, while expression of GRS1 was somewhat repressed under those conditions. In addition, GlyRS2 (the enzyme encoded by GRS2) had a higher protein stability and a lower K(M) value for yeast tRNA(Gly) under heat shock conditions than under normal conditions. Moreover, GRS2 rescued the growth defect of a GRS1 knockout strain when highly expressed by a strong promoter at 37 °C, but not at the optimal temperature of 30 °C. These results suggest that GRS2 is actually an inducible gene that may function to rescue the activity of GRS1 under stress conditions.

Figure 1. Comparison of Yeast GlyRS1 and GlyRS2.

(A) Alignment of the leader sequences of GlyRSs from various yeasts. A lowercase “m” denotes the initiating methionine encoded by the most promising non-AUG initiator candidate. For clarity, the initiating methionines of the mitochondrial and cytoplasmic forms of Saccharomyces cerevisiae GlyRS1 are each marked with an arrow above the sequence. (B) Sequence homology among yeast GlyRSs. 1, ScGlyRS1; 2, ScGlyRS2; 3, VpGlyRS1; and 4, VpGlyRS2. (C) Alignment of the lysine-rich insertion domains. Sc, Saccharomyces cerevisiae; Ca, Candida albicans; Yl, Yarrowia lipolytica; Sp, S. pombe; Ag, Ashbya gossypii; Cg, Candida glabrata; Cn, Cryptococcus neoformans; Dh, Debaryomyces hansenii; Kl, Kluyveromyces lactis; Le, Le, Lodderomyces elongisporus; Mg, Malassezia globosa; Pg, Pichia guilliermondii; Ps, Pichia stipitis; and Vp, Vanderwaltozyma polyspora.

Figure 2. Complementation Assays for the Yeast GRS2 Gene.

(A) Schematic summary of GRS1 and GRS2 constructs. GRS1 and GRS2 were individually cloned into pADH, and the ability of the constructs to rescue the growth defects of the knockout strain was tested. MTS, mitochondrial targeting sequence (amino acid residues 1∼46) of the mitochondrial precursor form of yeast ValRS. (B) Complementation assays for cytoplasmic (on FOA plates) and mitochondrial (on YPG plates) activities. Mit, mitochondrial; Cyt, cytoplasmic. (C) Western blot analysis. Aliquots of the protein extracts loaded onto the gel are shown under the blots. Upper panel, GlyRS; lower panel, phosphoglycerate kinase (PGK) (as a loading control). In (B–C), the numbers 1∼3 (circled) denote constructs shown in (A).

Figure 3. Relative Levels of GRS1 and GRS2 mRNAs.

(A) Semiquantitative RT-PCR. Relative levels of endogenous GRS1 and GRS2 mRNAs were determined by a semiquantitative RT-PCR (32 cycles). The primers used to amplify the GRS1 and GRS2 cDNA fragments are shown. Total RNA was isolated from cells grown under various culture conditions: normal conditions; growth temperature at 16°C; growth temperature at 37°C; growth medium at pH 8.0; growth medium with 3% ethanol; and growth medium with 5 mM H₂O₂. gDNA, genomic DNA; cDNA, complementary DNA. (B) Quantitative real-time RT-PCR. Primers used in the quantitative RT-PCR are described in “Materials and methods”.

Figure 4. Degradation Assays for GlyRS1 and GlyRS2.

Transformants were grown to a cell density of ∼1.0 A ₆₀₀, and then induced with galactose for 2 h before the addition of cycloheximide. Cells were harvested at various time points following treatment with cycloheximide and lysed. T₀, T₂, T₄, T₈, and T₁₂ respectively denote 0, 2, 4, 8, and 12 h post-induction. Upper panel, GlyRS; lower panel, phosphoglycerate kinase (PGK; as a loading control). Quantitative data for relative levels of GlyRSs are shown as a separate diagram below the Western blots.

Figure 5. Aminoacylation Activities of GlyRS1 and GlyRS2.

Aminoacylation activities of purified recombinant GlyRS-His₆ enzymes were determined at 30°C by measuring the relative amounts of ³H-glycine that were incorporated into tRNA using a liquid scintillation counter.