Import and processing of precursor to mitochondrial aspartate aminotransferase. Structure-function relationships of the presequence.
The precursor protein of pig mitochondrial aspartate aminotransferase (pre-mAspAT) contains a 29-residue presequence (Joh, T., Nomiyama, H., Maeda, S., Shimada, K., and Morino, Y. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1-5). Pre-mAspAT produced in an in vitro transcription and translation system was avidly imported into pig and rat liver mitochondria to be processed to the mature form of the enzyme. The pre-mAspAT was also processed to the mature form upon incubation with mitochondrial extracts. We synthesized precursor proteins with alterations within the presequence and compared quantitatively the effects of these mutations on the rates of both import and processing. Single and multiple substitutions of four basic residues with neutral amino acids at positions 5, 8, 18, and 28 showed that each residue contributes differentially to import and processing. Substitutions of His5 and Arg8 with glycines abolished the import activity but did not appreciably affect the rate of processing. Substitution of Arg28 with leucine at the position adjacent to the cleavage site seriously impaired the processing without appreciably affecting the rate of import. Analysis of deletions revealed that the amino-terminal region from position 2 to 8 was essential for both the import and processing. Thus the positive charges in the amino-terminal region are critical for import while the amino-terminal peptide segment and the cleavage site region appear to be requisite for recognition by a processing protease.
Nishi
T
T
Department of Biochemistry, Kumamoto University Medical School, Japan.
Nagashima
F
F
Tanase
S
S
Fukumoto
Y
Y
Joh
T
T
Shimada
K
K
Matsukado
Y
Y
Ushio
Y
Y
Morino
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001692:Biological Transport; D004252:DNA Mutational Analysis; D066298:In Vitro Techniques; D007526:Isoelectric Point; D008930:Mitochondria, Liver / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D013552:Swine; D013696:Temperature
D011498:Protein Precursors; D001219:Aspartate Aminotransferases