Import of mutant forms of mitochondrial aspartate aminotransferase into isolated mitochondria.
To gain some insight into the role played by certain protein domains in the import of mitochondrial aspartate aminotransferase in isolated mitochondria, three protein mutants were constructed by using the plasmid pOTS-mAspAT, which contains the nucleotide sequence encoding for the mature form of this enzyme. Two mutant proteins in which Cys-166 was substituted with either serine or alanine and another protein lacking the nine N-terminal amino acids were all synthesized in a cell-free transcription/translation system. Comparison was made among the newly synthesized mutant proteins and the newly synthesized wild type aspartate aminotransferase with respect to their capability to enter mitochondria. All the mutant proteins proved to be able to enter mitochondria even though with a lower efficiency than the wild type enzyme. Interestingly the thiol reagent mersalyl proved to inhibit import of both wild type enzyme and serine mutant, whereas import of alanine mutant was found to be insensitive to mersalyl, thus showing that Cys-166 is the unique -SH group involved in import. Import of mitochondrial aspartate aminotransferase by mitochondria is shown to involve certain protein domains present in the mature protein, two of them being the Cys-166 and the N-terminal regions.
Giannattasio
S
S
C.N.R. Centro di Studio sui Mitocondri e Metabolismo Energetico, Bari, Italy.
Marra
E
E
Vacca
R A
RA
Iannace
G
G
Quagliariello
E
E
D000595:Amino Acid Sequence; D001219:Aspartate Aminotransferases / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002474:Cell-Free System; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D015183:Restriction Mapping; D017384:Sequence Deletion
D009838:Oligodeoxyribonucleotides; D001219:Aspartate Aminotransferases