In vitro characterization of the mitochondrial processing and the potential function of the 68-kDa subunit of renal glutaminase

Abstract

Rat renal mitochondrial glutaminase (GA) is initially synthesized in primary cultures of proximal tubule cells as a 74-kDa precursor and is processed via a 72-kDa intermediate to generate a heterotetrameric enzyme which contains three 66-kDa subunits and one 68-kDa subunit (Perera, S. Y., Chen, T. C., and Curthoys, N. P. (1990) J. Biol. Chem. 265, 17764-17770). The two mature subunits may be derived by either of two possible mechanisms: 1) alternative proteolytic processing or 2) initial synthesis of the 66-kDa subunit followed by its covalent modification to generate the 68-kDa subunit. An in vitro system was utilized to further characterize this unique processing pathway and to investigate the potential function of the 68-kDa subunit. In vitro transcription and translation of the GA cDNA yields a single 74-kDa precursor. Upon incubation with isolated rat liver mitochondria, the precursor is translocated into the mitochondria and processed via a 72-kDa intermediate to yield a 3:1 ratio of the 66- and 68- kDa subunits, respectively. The kinetics of the in vitro processing reaction also closely approximate the kinetics observed in cultured cells. Mitochondrial processing is blocked by o-phenanthroline, an inhibitor of the matrix processing peptidase (MPP). The 72-amino acid presequence of the 66- kDa subunit contains a large proportion of basic amino acids. Two-dimensional gel electrophoresis of mature GA established that the 68-kDa subunit is slightly more basic than the 66-kDa subunit. In addition, incubation of the 74-kDa precursor with purified MPP yields equimolar amounts of the two mature peptides. A cDNA construct, pΔGA, was created which lacks the nucleotides that encode the amino acid residues 32 through 72 of GA. When transcribed and translated in vitro, pΔGA yields a 70-kDa precursor. This precursor is processed by mitochondria to a single mature subunit with a M of 66 kDa. This observation suggests that the 68-kDa subunit is not produced by covalent modification of the 66-kDa subunit and further supports the conclusion that the two mature subunits of GA are produced by alternative processing reactions which can be catalyzed by MPP. However, the yield of products obtained in intact mitochondria may be determined by some unidentified accessory factor. Submitochondrial fractionation of imported GA and ΔGA precursors suggest that the 68-kDa subunit may function to retain the mature GA within the mitochondrial matrix.