Biochemical comparison of the Neurospora crassa wild type and the temperature‐sensitive and leucine‐auxotroph mutant leu‐5

Abstract

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 "C) and mutant (grown at 28 "C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28°C and transferred then to 37°C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37°C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish any difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 "C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of-1 : 600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme. The mutant leu-5 of Neurospora crassa, which is temperature sensitive and leucine-auxotroph, has been reported earlier to have a mutation in the leucyl-tRNA synthetase gene, providing a cytoplasmic leucyl-tRNA synthetase with an increased K, for leucine, a protein biosynthesis with one or some inaccurate steps and very little mitochondrial leucyl-tRNA synthetase [ 11. These phenomena have been tentatively explained by Gross et al. in a proposal regarding the structure and function of the corresponding genes [2-41. Enzyme. Leucyl-tRNA synthetase (EC 6.1.1.4). However, the molecular biology of the mutation has not been established at the DNA level nor at the protein level, and this will be of interest regarding the structure and function of aminoacyl-tRNA synthetases. Moreover a leucyl-tRNA synthetase with reduced function may establish th...

Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

“…These effects were interpreted as being due to misaminoacylation of cytoplasmic tRNAs in the leu-S strain by a cytoplasmic LeuRS having an increased Km for leucine, resulting in misincorporation during cytoplasmic protein synthesis. However, the alteration in the Km value for leucine of the cytoplasmic LeuRS seen in crude extracts (3) could not be reproduced with purified enzyme (35). Since we have shown here that the defect in leu-5 is in the mitochondrial LeuRS gene, an alternative explanation is that in an obligate aerobe such as N. crassa, defects in mitochondrial protein synthesis could have other effects which influence the stability of proteins in extracts.…”

“…Having shown that the cytoplasmic LeuRS structural gene did not map to the leu-5 locus (4), we wanted to isolate the mitochondrial LeuRS structural gene and determine its relationship to leu-5. The leu-5 mutant has virtually no detectable mitochondrial LeuRS activity (1,35,62); however, concomitant alterations in several other enzymes, including the cytoplasmic LeuRS, were reported (38,49). Consequently, it was not known whether the mutation in leu-5 was in the mitochondrial LeuRS structural gene or some other gene.…”

“…However, using hybrid mitochondrial LeuRS genes, we localized the mutation responsible for the leu-5 phenotype to a 314-bp fragment (Fig. 7) (35,49; data not shown), suggests that the Thr-to-Pro missense mutation results in a temperature-sensitive protein, which is not stable to extraction or in vitro assays. Such behavior is observed for many temperaturesensitive proteins.…”

“…7, N. crassa is an obligate aerobe; therefore, the mitochondrial LeuRS gene is expected to be essential. Although leu-5 was reported to lack detectable mitochondrial LeuRS activity in vitro even when grown under permissive conditions (35,62; data not shown), some activity must exist in vivo to allow growth of the strain. For example, mitochondria from leu-5 grown under permissive conditions show normal cytochrome spectra (1).…”