| dc.description.abstract | It was previously shown that the cytoplasmic and mitochondrial activities of
alanyl-tRNA synthetase of Saccharomyces cerevisiae are provided by two distinct translational
products of ALA1, one initiated at the AUG codon closest to the 5’-end of its mRNA
transcripts and the other at upstream in-frame redundant non-AUG codons (i.e., ACG-25 and
ACG-24). We report here the cloning and characterization of the only alanyl-tRNA synthetase
gene of Candida albicans (designated as CaALA1). Cross-species complementation assays
suggest that CaALA1 can substitute for both the cytoplasmic and mitochondrial functions of
ALA1 in S. cerevisiae. However, unlike the case in S. cerevisiae, both of these two activities are
provided a single primary translational product of CaALA1 that is initiated from the first AUG
codon (designated as AUG1) on its coding sequence. When the first AUG codon is inactivated
by point mutations, the mitochondrial function of this gene is impaired, presumably due to
inability to initiate protein synthesis, while its cytoplasmic function can be rescued by
alternative initiation at a downstream AUG codon, AUG9. Functional mapping using the
cytoplasmic form of valyl-tRNA synthetase as the passenger protein identifies the first 42
amino-terminal residues of the Candida protein as the mitochondrial targeting signal. In
addition, evidence shows that an exclusive mitochondrial targeting signal is translationally
initiated from the first AUG initiator.
In the second part of this thesis, we focus on the transport mechanism and physiological
function of mitochondrial ScAlaRS. The results of functional tests, site-directed mutagenesis,
and western blotting all suggest that the mitochondrial targeting signal of ScAlaRS includes
not only the leader peptide but also the first eighteen amino acids downstream of Met1. Neither
the leader peptide nor the eighteen-residue peptide can function as a transit signal per se. By
western blotting assays, our data shows that the ACG-25-initiated proteins are fully translocated
into the mitochondria, and the AUG1-initiated proteins are almost completely retained in the
cytosol. These results confirm our previous observations that cytoplasmic and mitochondrial
activities of ScALA1 are provided by two distinct translational products. In addition, partition
of ScAlaRS is determined by both its leader peptide and expression levels. | en_US |