Biomedical subjects

O Gakh

Publications and source records attributed to O Gakh.

3 recordsLinked to original sources

PubMed2001

Substrate binding changes conformation of the alpha-, but not the beta-subunit of mitochondrial processing peptidase.

Lifetime analysis of tryptophan fluorescence of the mitochondrial processing peptidase (MPP) from Saccharomyces cerevisiae clearly proved that substrate binding evoked a conformational change of the alpha-subunit while presence of substrate influenced neither the lifetime components nor the average lifetime of the tryptophan excited state of the beta-MPP subunit. Interestingly, lifetime analysis of tryptophan fluorescence decay of the alpha-MPP subunit revealed about 11% of steady-state fractional intensity due to the long-lived lifetime component, indicating that at least one tryptophan residue is partly buried at the hydrophobic microenvironment. Computer modeling, however, predicted none of three tryptophans, which the alpha-subunit contains, as deeply buried in the protein matrix. We conclude this as a consequence of a possible dimeric (oligomeric) structure.

Amino Acid Sequence↗

PubMed2000

Two-step processing of human frataxin by mitochondrial processing peptidase. Precursor and intermediate forms are cleaved at different rates.

We showed previously that maturation of the human frataxin precursor (p-fxn) involves two cleavages by the mitochondrial processing peptidase (MPP). This observation was not confirmed by another group, however, who reported only one cleavage. Here, we demonstrate conclusively that MPP cleaves p-fxn in two sequential steps, yielding a 18,826-Da intermediate (i-fxn) and a 17,255-Da mature (m-fxn) form, the latter corresponding to endogenous frataxin in human tissues. The two cleavages occur between residues 41-42 and 55-56, and both match the MPP consensus sequence RX downward arrow (X/S). Recombinant rat and yeast MPP catalyze the p --> i step 4 and 40 times faster, respectively, than the i --> m step. In isolated rat mitochondria, p-fxn undergoes a sequence of cleavages, p --> i --> m --> d(1) --> d(2), with d(1) and d(2) representing two C-terminal fragments of m-fxn produced by an unknown protease. The i --> m step is limiting, and the overall rate of p --> i --> m does not exceed the rate of m --> d(1) --> d(2), such that the levels of m-fxn do not change during incubations as long as 3 h. Inhibition of the i --> m step by a disease-causing frataxin mutation (W173G) leads to nonspecific degradation of i-fxn. Thus, the second of the two processing steps catalyzed by MPP limits the levels of mature frataxin within mitochondria.

Amino Acid Sequence↗

PubMed1999

Complementation between mitochondrial processing peptidase (MPP) subunits from different species.

Mitochondrial processing peptidase (MPP), a dimer of nonidentical subunits, is the primary peptidase responsible for the removal of leader peptides from nuclearly encoded mitochondrial proteins. Alignments of the alpha and beta subunits of MPP (alpha- and beta-MPP) from different species show strong protein sequence similarity in certain regions, including a highly negatively charged region as well as a domain containing a putative metal ion binding site. In this report, we describe experiments in which we combine the subunits of MPP from yeast, rat, and Neurospora crassa, both in vivo and in vitro and mesure the resultant processing activity. For in vivo complementation, we used the temperature sensitive mif1 and mif2 yeast mutants, which lack MPP activity at the nonpermissive temperature (37 degrees C). We found that the defective alpha-MPP of mif2 cannot be substituted for by the alpha-MPP from rat or Neurospora. On the other hand, the beta-MPP from rat and Neurospora can fully substitute for the defective beta-MPP in the mif1 mutant. These results were confirmed in in vitro experiments in which individually expressed subunits were combined. Only combinations of the alpha-MPP from yeast with the beta-MPP from rat or Neurospora produced active MPP.

Animals↗