Suppressive effect of the overexpression of NAM2 and its variants. (A) Serial dilutions of WT (MCC123), of the isogenic LeuC25T mutant and of the same mutant transformed with multicopy empty plasmid and multicopy plasmids bearing the WT NAM2 gene (coding for Sc mt leucyl-tRNA synthetase) or its different variants obtained by deletion or isolation of individual domains as indicated. The tested domains are the CP1 (Connecting Peptide 1 having editing function) and the C-terminal domains. All strains were spotted on YP plate containing 3% glycerol and incubated at 28 °C. The same results were obtained at 37 °C. (B) Graphic representation of the results shown in panel A. Values obtained by Phoretix analysis are calculated as percentage of the WT.

Suppressive effect of aaRS and parts thereof. (A) Serial dilutions of WT (MCC123), of the isogenic ValC25T mutant, of the mutant transformed with multicopy empty plasmid and multicopy plasmids bearing the VAS1 gene (coding for Sc mt ValRS), the NAM2 gene (coding for Sc mt LeuRS), the LARS2 gene (coding for Hs mt LeuRS), two CtermLARS2 variants (30_31 and 32_33) and the GFP gene fused to the mt pre-sequence of ATPase subunit 9 of Neurospora crassa (pmtGFP), as control. The strains are spotted on a YP plate containing 3% glycerol and incubated at 28 °C for three days. The same results were obtained at 37 °C. (B) Serial dilutions of the WT (MCC123), of the thermosensitive IleT33C mutant, of the same mutant transformed with multicopy empty plasmid or multicopy plasmids bearing ISM1 gene (coding for the Sc mt Isoleucyl-tRNA synthetase), and other sequences as in panel A were spotted on YP plate containing 3% glycerol and incubated at 37 °C for three days.

Suppressive effect of the CtermNAM2 domain. (A) Growth phenotype of the WT (MCC123), of isogenic mt tRNA mutants (Val, Leu and Ile), and of the same mutants transformed with the C-terminal domain of LeuRS sequence cloned into the multicopy vector (pCtermNAM2) under the inducible GAL1 promoter. The strains were plated on YP 3% glycerol and YP 3% glycerol with addition of 0.1% galactose containing media and grown for three days at 28 °C. (B) Multiple sequence alignment of the C-terminal domain of mt LeuRS (H. sapiens, Hs, S. cerevisiae, Sc, and T. thermophilus, Tt). Identical aminoacids are highlighted in black; the aminoacids with similar charge are highlighted in gray. The secondary structure elements are indicated by helices (α-helices) and arrows (β-strands). Subcloned sequences contacting the tRNA elbow (http://www.expasy.org/spbdv/) are boxed.

Cellular localization of LeuRS and its variants. (A) Fluorescence microscopy of ValC25T (upper panels) and LeuC25T (lower panels) mutants transformed with pNAM2GFP and pCtermNAM2GFP. The GFP gene sequence was cloned in frame at the 3′-end of NAM2 sequences. As control (bottom panel) the GFP YEAST CLONE (Invitrogen) with the endogenous NAM2-GFP fusion. (B) Fluorescence microscopy of WT (MCC123), ValC25T and LeuC25T transformed with a multicopy plasmid bearing the GFP gene with the mt pre-sequence of ATPase subunit 9 of N. crassa fused at 5′-end (pmtGFP).