FUNCTIONAL COOPERATION OF THE MITOCHONDRIAL PROCESSING PEPTIDASE SUBUNITS

Domains important for the activity of the heterodimeric mitochondrial processing peptidase (MPP) were investigated, by inserting one alanine residue at ten positions along the polypeptide chain of the beta-subu nit (beta-MPP). An alanine residue inserted after Glu70, Ser114, Lys21 5 and Ser314 respectively, abolished the cleavage activity of MPP. Whe n the alpha-subunit (alpha-MPP) was co-expressed with N-terminal hexa- histidine tagged beta-MPP, alpha-MPP was co-eluted from a nickel-deriv atized affinity resin, with a 1:1 stochiometry, both with wild-type be ta-MPP and with the mutants with alanine inserted after Ser114 and Ser 314. The mutants with alanine inserted after Glu70 and Lys215 did not associate with alpha-MPP. The mutagenesis studies indicate that: (1) t he whole HXXEHX76H region of beta-MPP is important for the proper conf ormation of the active site of MPP and may also be in contact with alp ha-MPP; (2) the non-conserved central region surrounding Lys215 is inv olved in the interaction with alpha-MPP; and (3) the carboxy-terminal region of beta-MPP surrounding Ser314 is also of importance for the ca talysis. Cross-linking studies indicated that purified alpha-MPP bound a precursor protein in the absence of any beta-MPP. Futhermore, the i nteraction of MPP and its subunits with a peptide substrate, as analyz ed by surface plasmon resonance, showed that alpha-MPP bound a peptide substrate as efficiently as MPP. The data suggest that the alpha-subu nit is responsible for the binding of mitochondrial presequences prior their presentation to the catalytic site of MPP. (C) 1997 Academic Pr ess Limited.