Presequence does not prevent folding of a purified mitochondrial precursor protein and is essential for association with a reticulocyte cytosolic factor(s).
Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) [EC 2.1.3.3] is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues, then is imported posttranslationally nto the mitochondrial matrix. We expressed rat pOTC in Escherichia coli, purified it in a denatured form, and showed that 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 properties 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.HCl, was essentially homogeneous, as judged by SDS-PAGE. When guanidine.HCl-denatured mature OTC was diluted and incubated at 0 degrees C for 40-60 h, it was reactivated to a specific activity of 170 mumol/min/mg protein at 37 degrees C (18% of that of the purified mature enzyme). Guanidine.HCl-denatured pOTC was activated to a specific activity of 125 mumol/min/mg protein under similar conditions. The native and reactivated OTC sedimented with an s20.w value of 6.2S, whereas the activated pOTC sedimented with an s20.w of 5.2S. The activated pOTC was more unstable than the reactivated OTC at 50 degrees C. 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Murakami
K
K
Institute for Medical Genetics, Kumamoto University Medical School.
Tokunaga
F
F
Iwanaga
S
S
Mori
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002499:Centrifugation, Density Gradient; D003600:Cytosol / Q000737:chemistry*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D019791:Guanidine; D006146:Guanidines / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011189:Potassium Chloride / Q000494:pharmacology; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012156:Reticulocytes / Q000737:chemistry*; D013395:Sucrose; D013462:Sulfur Radioisotopes
D004792:Enzyme Precursors; D006146:Guanidines; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D013395:Sucrose; D011189:Potassium Chloride; D009954:Ornithine Carbamoyltransferase; D019791:Guanidine