Multiple Quality Control Pathways Limit Non-protein Amino Acid Use by Yeast Cytoplasmic Phenylalanyl-tRNA Synthetase

Abstract

Non-protein amino acids, particularly isomers of the proteinogenic amino acids, present a threat to proteome integrity if they are mistakenly inserted into proteins. Quality control during aminoacyl-tRNA synthesis reduces non-protein amino acid incorporation by both substrate discrimination and proofreading. For example phenylalanyl-tRNA synthetase (PheRS) proofreads the non-protein hydroxylated phenylalanine derivative m-Tyr after its attachment to tRNA(Phe) We now show in Saccharomyces cerevisiae that PheRS misacylation of tRNA(Phe) with the more abundant Phe oxidation product o-Tyr is limited by kinetic discrimination against o-Tyr-AMP in the transfer step followed by o-Tyr-AMP release from the synthetic active site. This selective rejection of a non-protein aminoacyl-adenylate is in addition to known kinetic discrimination against certain non-cognates in the activation step as well as catalytic hydrolysis of mispaired aminoacyl-tRNA(Phe) species. We also report an unexpected resistance to cytotoxicity by a S. cerevisiae mutant with ablated post-transfer editing activity when supplemented with o-Tyr, cognate Phe, or Ala, the latter of which is not a substrate for activation by this enzyme. Our phenotypic, metabolomic, and kinetic analyses indicate at least three modes of discrimination against non-protein amino acids by S. cerevisiae PheRS and support a non-canonical role for SccytoPheRS post-transfer editing in response to amino acid stress.

Keywords: Quality control; amino acid; aminoacyl tRNA synthetase; protein synthesis; transfer RNA (tRNA); translation.

FIGURE 1.

Potential non-cognate amino acid substrates of PheRS. Shown are hydroxylated phenylalanine derivatives that may accumulate as the result of oxidative damage, metabolic conversion, or import.

FIGURE 2.

SccytoPheRS post-transfer editing limited the cytotoxicity of oxidative stress. Growth of wild-type (wt) or post-transfer editing ablated SccytoPheRS (frs1−1) grown in minimal media with or without 2.0 mm paraquat. Data represent the average of measurements from three cultures. Error bars represent ± S.E.

FIGURE 3.

Oxidative stress increased the abundance of non-protein amino acids. Abundance of extracted intracellular amino acids from 10-ml samples of exponentially growing wild-type or frs1−1 grown in minimal media with or without 2.0 mm paraquat. Values are normalized to the optical density at 600 nm of parent cultures at the time of harvest. Samples were taken from triplicate cultures. Error bars represent ± S.E. *, p < 0.05, unpaired t test. One of the three biological samples taken had a total metabolite mass ∼2.5–2.8 times that of the other two replicates, resulting in sufficient variability to not meet the standard conventions of statistical significance for the comparison between unstressed and stressed wild-type cultures for p-Tyr (p = 0.0581), m-Tyr (p = 0.1455), and l-DOPA (p = 0.1127). There was also no significant difference between the amount of m-Tyr in frs1−1 with or without paraquat (p = 0.1867).

FIGURE 4.

SccytoPheRS post-transfer editing activity had variable effects on growth rate dependent on supplemented amino acids. Exponential phase growth rate of wild-type (dark gray) and frs1−1 (light gray) yeast in minimal media supplemented with indicated amino acids. Data represent the average of measurements from three cultures. Error bars represent ± S.E. *, p < 0.05, unpaired t test.

FIGURE 5.

o-Tyr was inefficiently acylated to tRNA^Phe. Aminoacylation of tRNA^Phe by post-transfer editing ablated SccytoPheRS with 75 μm Phe, 75 μm p-Tyr, and 150 μm dl-o-Tyr, expressed as fraction chargeable tRNA^Phe ± S.E. (n = 3). tRNA^Phe was transcribed in vitro without A76 before addition of A76 with tRNA nucleotidyltransferase using α-[³²P]ATP.

FIGURE 6.

o-Tyr-AMP was not efficiently hydrolyzed by SccytoPheRS. o-Tyr-AMP accumulates to 17-fold greater than wild-type (panel A) or βD243A (panel B) SccytoPheRS by 2 min. AMP is not appreciably formed. Data are presented as the average of three replicates ± S.E. o-Tyr-AMP is stable in the absence of SccytoPheRS under reaction conditions (supplemental Fig. 3).