Efficient but aberrant cleavage of mitochondrial precursor proteins by the chloroplast stromal processing peptidase.

Cytosol-synthesized chloroplast and mitochondrial precursor proteins are proteolytically processed after import by highly specific, metal-dependent soluble enzymes: the stromal processing peptidase (SPP) and the matrix processing peptidase (MPP), respectively. We have used in vitro processing assays to compare the reaction specificities of highly purified preparations of pea SPP and Neurospora crassa MPP, both of which are unable to cleave a variety of 'foreign' proteins. We show that SPP can cleave all five mitochondrial precursor proteins tested, namely cyclophilin, the beta subunit of the F1-ATPase complex, the Rieske FeS protein, the alpha-MPP subunit and cytochrome b2. In contrast, MPP is unable to cleave any chloroplast precursor proteins tested. Several of the mitochondrial precursor proteins are cleaved more efficiently by SPP than are many authentic chloroplast precursor proteins but, in each case, cleavage takes place at a site or sites which are N-terminal to the authentic MPP site; pre-cyclophilin is cleaved 5 residues upstream of the MPP site and the precursor of the beta subunit of the F1-ATPase complex is cleaved at sites 5 and 12 residues upstream. We discuss the implications of these data for the SPP reaction mechanism.

Spontaneous dissociation of a cytochrome core and a biglobular flavoprotein after mild trypsinolysis of the bifunctional Saccharomyces cerevisiae flavocytochrome b2.

Saccharomyces cerevisiae flavocytochrome b2 is known as a bifunctional enzyme which behaves as the association of an FMN flavodehydrogenase with its specific acceptor, a b5-like cytochrome. Mild trypsinolysis gives rise to three complementary fragments (n, X, beta'), both prosthetic groups being still bound. After such proteolysis the separation of a biglobular flavoprotein domain (carrying FMN) from a cytochrome domain (with the heme) is obtained by molecular sieving under non-denaturing conditions. The marked lack of affinity between the tetrameric flavoprotein (X, beta')4 and the monomeric cytochrome core (n) leads to the hypothesis that the two domains are not tightly associated in the native molecule and might more relative to each other. Their respective mobility is possibly required for the catalytic mechanism. The comparison with previous trypsinolysis studies on the flavocytochrome b2 from Hansenula anomala suggests the presence of two common zones of hypersensitivity to proteases, along the protomeric polypeptide chain, and strongly supports the validity of the triglobular model for both flavocytochromes.