J Biol Chem, Vol. 273, Issue 14, 7972-7980, April 3, 1998

Processing and Function of a Polyprotein Precursor of Two Mitochondrial Proteins in Neurospora crassa

From the Department of Chemistry and Biochemistry, UCLA, Los Angeles, California 90095-1569

In Neurospora crassa, the mitochondrial arginine biosynthetic enzymes, N-acetylglutamate kinase (AGK) and N-acetyl--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-GlyTyr-Leu-Thr at the N terminus of the AGPR domain and Arg-Gly-TyrSer-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 AGKAGPR mutants. These results indicate that cleavage of pAGK-AGPR is not required for functioning of these enzymes in the mitochondria.