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 cy tosolic precursor that is translocated into mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of mature 66- and 68-k Da subunits, respectively. The 66 kDa subunit is derived by removal of a 72-amino-acid presequence, The structural determinants necessary fo r 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 precur sor to chloramphenicol acetyl transferase (CAT). GA(1-118)CAT is trans located and processed in isolated rat liver mitochondria or cleaved by purified mitochondrial processing peptidase (MPP) to yield an interme diate peptide and two mature subunits that are analogous to the produc ts of processing of the GA precursor. The two reactions also occur wit h kinetics which are similar to those observed for processing of the G A precursor, Thus, all of the information required for the translocati on and synthesis of the mature subunits of GA reside in the N-terminal 118 amino acids of the GA precursor. In contrast, GA(1-72)CAT, a cons truct that contains the GA presequence fused to CAT, is apparently tra nslocated and processed less efficiently. It yields only two peptides that are analogous to the intermediate and 68 kDa forms of GA. In addi tion, GA(1-31)CAT associates with mitochondria but is not proteolytica lly processed and GA(1-31,73-118)CAT is slowly translocated and proces sed to a single peptide that is analogous to the 66 kDa form of GA, Th e latter results suggest that the MPP cleavage reactions which yield t he GA intermediate and the 66 kDa subunit depend primarily on informat ion that is present C-terminal to the respective sites of cleavage.