J. Biochem, 1990, Vol. 108, No. 2 207-214
© 1990 Japanese Biochemical Society
Presequence Does Not Prevent Folding of a Purified Mitochondrial Precursor Protein and Is Essential for Association with a Reticulocyte Cytosolic Factor(s)1
Kaoru Murakami*,
Fuminori Tokunaga**,***,
Sadaaki Iwanaga*** and
Masataka Mori*
*Institute for Medical Genetics, Kwnamoto University Medical School Kumarnoto, Kumamoto 862
**Department of Molecular Biology, Graduate School of Medical Science, Kyushu University Higashi-ku, Fukuokij, Fukuoka 812
***Department of Biology, Faculty of Science, Kyushu University Higashi-ku, Fukuokij, Fukuoka 812
Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) [EC 2.1.3.3] is initially synthesized as a precursor (pOTC) with a transient NB2-terminal presequence of 32 amino acid residues, then is imported posttranslationally into the mitochondrial matrix. We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that it could be transported into isolated mitochondria in the presence of rabbit reticulocyte lysate [Murakami et al. (1988) J. Biol. Chem. 263, 18437–18442]. In order to compare the prop erties of the precursor and mature form of OTC, the rat mature OTC was synthesized in E. coli and purified. The recombinant OTC represented about 5% of the total bacterial protein and was present in both the supernatant and precipitate of the disrupted bacteria. The OTC, extracted from the precipitate with 8 M urea or 6 M guanidine was essentially homogeneous, as judged by SDS-PAGE. When guanidine.HC1-denatured mature OTC was diluted and incubated at 0°C for 40–60 h, it was reactivated to a specific activity of 170 µmol/ min/mg protein at 37°C (18% of that of the purified mature enzyme). Guanidine.HC1-denatured OTC was activated to a specific activity of 125 µmol/min/mg protein under similar conditions. The native and reactivated OTC sedimented with an s value of 6.28, whereas the activated pOTC sedimented with an 820.w of 5.2S. The activated pOTC was more unstable than the reactivated OTC at 50C. These observations indicate that the presequence does not prevent pOTC from folding into an enzymatically active trimeric form, although the pOTC trimer appears to be less compact than the mature trimer. Sucrose gradient centrifugation analysis showed that pOTC, but not mature OTC, formed a complex sedimenting with an & values of 6-uS in the presence of reticulocyte lysate. Formation of the complex of 35 pOTC was inhibited by unlabeled pOTC or synthetic prese quence but not by mature OTC. These observations taken together with our previous findings suggest that a protein factor(s) present in the reticulocyte lysate intracts with the presequence portion of pOTC, prevents pOTC from folding, and holds it in an import- competent form.
1This work was supported in part by a Grant-in-Aid (6348130) from the Ministry of Education, Science and Culture of Japan, a grant from Nissan Science Foundation and a grant from the Inamori Foundation.
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