Role of the N-terminal 118 amino acids in the processing of the rat renal mitochondrial glutaminase precursor.
Rat renal mitochondrial glutaminase (GA) is synthesized as a 74-kDa cytosolic precursor that is translocated into mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of mature 66- and 68-kDa subunits, respectively. The 66-kDa subunit is derived by removal of a 72-amino-acid presequence. The structural determinants necessary for translocation and proteolytic processing were further delineated by characterizing the processing of different chimeric constructs formed by fusing various segments of the N-terminal sequence of the GA precursor to chloramphenicol acetyl transferase (CAT). GA1-118 CAT is translocated and processed in isolated rat liver mitochondria or cleaved by purified mitochondrial processing peptidase (MPP) to yield an intermediate peptide and two mature subunits that are analogous to the products of processing of the GA precursor. The two reactions also occur with kinetics which are similar to those observed for processing of the GA precursor. Thus, all of the information required for the translocation and synthesis of the mature subunits of GA reside in the N-terminal 118 amino acids of the GA precursor. In contrast, GA1-72 CAT, a construct that contains the GA presequence fused to CAT, is apparently translocated and processed less efficiently. It yields only two peptides that are analogous to the intermediate and 68 kDa forms of GA. In addition, GA1-31 CAT associates with mitochondria but is not proteolytically processed and GA1-31,73-118 CAT is slowly translocated and processed to a single peptide that is analogous to the 66 kDa form of GA. The latter results suggest that the MPP cleavage reactions which yield the GA intermediate and the 66-kDa subunit depend primarily on information that is present C-terminal to the respective sites of cleavage.
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins 80523.
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Srinivasan
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Kalousek
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Farrell
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Curthoys
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism*; D005972:Glutaminase / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008297:Male; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D000596:Amino Acids; D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D005972:Glutaminase