Biosynthesis and processing of renal mitochondrial glutaminase in cultured proximal tubular epithelial cells and in isolated mitochondria.
Primary cultures of rat renal proximal tubular epithelial cells were used to characterize the biosynthesis and processing of the mitochondrial glutaminase. When the cells were labeled with [35S]methionine in the presence of 20 microM carbonylcyanide m-chlorophenylhydrazone, only a 72-kDa peptide, which co-migrates with the primary translation product of the glutaminase mRNA, was immunoprecipitated. At lower concentrations of carbonylcyanide m-chlorophenylhydrazone, the 68- and 65-kDa peptides that are characteristic of the mature glutaminase and a 71-kDa peptide were synthesized. Pulse-chase experiments suggest that the 72-kDa cytosolic precursor could be quantitatively chased to generate the mature mitochondrial species. The observed kinetics indicate that the 71-kDa species is an intermediate in the import pathway. In addition, the 65-kDa glutaminase peptide was synthesized more rapidly than the 68-kDa peptide, and the two peptides were produced in a final ratio of 3:1, respectively. These results suggest that one subunit of the tetrameric glutaminase may be subject to covalent modification. In vitro processing was also characterized by incubating isolated rat liver mitochondria with the glutaminase precursor that was produced by in vitro translation of acidotic rat renal poly(A+) RNA. This system produced an identical sequence of processing reactions. The in vitro formation of the 71-kDa intermediate required a transmembrane potential. Both the intermediate and the mature forms of the glutaminase were recovered in the mitochondria and were resistant to trypsin digestion. Thus, the glutaminase precursor is rapidly translocated across the inner mitochondrial membrane and initially processed to yield an intermediate. The intermediate is subsequently processed to yield the two peptides that constitute the mature enzyme.
Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
S Y
SY
Perera
T C
TC
Chen
N P
NP
Curthoys
D000818:Animals; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004848:Epithelium / Q000201:enzymology; D005972:Glutaminase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007672:Kidney Cortex / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008928:Mitochondria / Q000201:enzymology*; D014176:Protein Biosynthesis / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains
D012333:RNA, Messenger; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D003513:Cycloheximide; D005972:Glutaminase