Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.
Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.
Ellis
S R
SR
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
Hopper
A K
AK
Martin
N C
NC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D015398:Signal Transduction; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D004271:DNA, Fungal; C059863:N(2),N(2)-dimethylguanosine-specific tRNA methyltransferase; D012359:tRNA Methyltransferases