To ascertain that translation of these two ALA1 mutants was actually initiated

from CGC or CAC, and not from other remedial initiation sites, codons in the leader sequence that have the potential to serve as secondary translation initiation sites and initiate the synthesis of at least part of the mitochondrial targeting sequence were targeted for mutagenesis, and the protein expression and complementation activity of the resultant mutants were then tested. In this regard, TTG(-16) appeared to be a promising candidate on account of its favorable sequence context. To distinguish the protein forms initiated from ACG(-25) and UUG(-16), an 18% polyacrylamide gel was used. As shown in Figure 3, mutation of ACG(-25) to CGC had only a minor effect on mitochondrial activity, but drastically reduced protein expression Bafilomycin A1 manufacturer (Figure 3A, B, numbers

CDK inhibitor drugs 1 and 2). The upper and lower protein bands were abolished by the mutation, while the middle band was largely unaffected. This result suggests that both the upper and lower bands were initiated from ACG(-25), and the lower band was derived from cleavage of the upper band possibly by a matrix-processing peptidase. A further mutation that changed TTG(-16) to TTA impaired both the mitochondrial activity and protein expression of the CGC mutant (Figure 3A, B, numbers 2 and 4), suggesting that UUG(-16) served as a remedial initiation

site in the CGC mutant and the middle band was initiated from UUG(-16). As the UUG codon possesses stronger initiating activity in the CGC mutant than in the GGU mutant (Figure 3B, numbers 2 and 3), it is possible that CGC(-25) rescued the initiating activity of UUG(-16). Note that the TTG-to-TTA change is a silent mutation and therefore does not affect the stability of the protein form initiated from ACG(-25). A semiquantitative RT-PCR experiment Axenfeld syndrome further demonstrated that these mutations at codon position -25 or -16 did not affect the stability of the mRNAs derived from these constructs (Figure 3C). Figure 3 Rescuing a cryptic translation initiation site in ALA1. (A) Complementation assays for mitochondrial AlaRS activity. (B) Assay of initiating activity by Western blots. Upper panel, AlaRS-LexA fusion; lower panel, PGK (as loading controls). (C) RT-PCR. Relative amounts of specific ALA1-lexA mRNAs generated from each construct were determined by RT-PCR. As a control, relative amounts of actin mRNAs were also determined. The ALA1 sequences used in the ALA1-lexA constructs 1~4 in (B) were respectively transferred from constructs 1~4 shown in (A). In (C) the numbers 1~4 (circled) denote constructs shown in (B).