Processing and function of a polyprotein precursor of two mitochondrial proteins in Neurospora crassa

• Published in: The Journal of biological chemistry Vol. 273; no. 14; pp. 7972 - 7980
• Main Authors: Parra-Gessert, L. (UCLA, Los Angeles, CA.), Koo, K, Fajardo, J, Weiss, R.L
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 03.04.1998
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALDEHYDE OXIDOREDUCTASES; Amino Acid Sequence; AMINO ACID SEQUENCES; CHEMICAL COMPOSITION; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; ENZYME INHIBITORS; ENZYME PRECURSORS; ENZYMIC ACTIVITY; Fungal Proteins - metabolism; INHIBIDORES DE ENZIMAS; INHIBITEUR D'ENZYME; KINASES; METABOLISME DES PROTEINES; METABOLISMO PROTEICO; MITOCHONDRIA; Mitochondria - metabolism; MITOCHONDRIE; MITOCONDRIA; MOLECULAR CONFORMATION; Molecular Sequence Data; MUTANT; MUTANTES; MUTANTS; N-ACETYL-GAMMA-GLUTAMYL-PHOSPHATE REDUCTASE; N-ACETYLGLUTAMATE KINASE; NEUROSPORA CRASSA; Neurospora crassa - metabolism; OXIDOREDUCTASES; OXIDORREDUCTASAS; OXYDOREDUCTASE; PEPTIDASAS; PEPTIDASE; PEPTIDASES; PRECURSEUR D'ENZYME; PRECURSORES DE ENZIMAS; PROTEIN METABOLISM; Protein Precursors - metabolism; Protein Processing, Post-Translational; Protein Structure, Secondary; PROTEIN TRANSPORT; PROTEOLISIS; PROTEOLYSE; PROTEOLYSIS; SECONDARY STRUCTURE; TRANSFERASAS; TRANSFERASE; TRANSFERASES
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.14.7972

In Neurospora crassa , the mitochondrial arginine biosynthetic enzymes, N -acetylglutamate kinase (AGK) and N -acetyl-γ-glutamyl-phosphate reductase (AGPR), are generated by processing of a 96-kDa cytosolic polyprotein precursor (pAGK-AGPR). The proximal kinase and distal reductase domains are separated by a short connector region. Substitutions of arginines at positions −2 and −3 upstream of the N terminus of the AGPR domain or replacement of threonine at position +3 in the mature AGPR domain revealed a second processing site at position −20. Substitution of arginine at position −22, in combination with changes at −2 and −3, prevented cleavage of the precursor and identified two proteolytic cleavage sites, Arg-Gly↓Tyr-Leu-Thr at the N terminus of the AGPR domain and Arg-Gly-Tyr↓Ser-Thr located 20 residues upstream. Inhibitors of metal-dependent peptidases blocked proteolytic cleavage at both sites. Amino acid residues required for proteolytic cleavage in the connector were identified, and processing was abolished by mutations changing these residues. The unprocessed AGK-AGPR fusion had both catalytic activities, including feedback inhibition of AGK, and complemented AGK − AGPR − mutants. These results indicate that cleavage of pAGK-AGPR is not required for functioning of these enzymes in the mitochondria.