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-subunit (beta-MPP). An alanine residue inserted after Glu70, Ser114, Lys215 and Ser314 respectively, abolished the cleavage activity of MPP. When the alpha-subunit (alpha-MPP) was co-expressed with N-terminal hexa-histidine tagged beta-MPP, alpha-MPP was co-eluted from a nickel-derivatized affinity resin, with a 1:1 stochiometry, both with wild-type beta-MPP and with the mutants with alanine inserted after Ser114 and Ser314. The mutants with alanine inserted after Glu70 and Lys215 did not associate with alpha-MPP. The mutagenesis studies indicate that: (1) the whole HXXEHX76H region of beta-MPP is important for the proper conformation of the active site of MPP and may also be in contact with alpha-MPP; (2) the non-conserved central region surrounding Lys215 is involved in the interaction with alpha-MPP; and (3) the carboxy-terminal region of beta-MPP surrounding Ser314 is also of importance for the catalysis. Cross-linking studies indicated that purified alpha-MPP bound a precursor protein in the absence of any beta-MPP. Furthermore, the interaction of MPP and its subunits with a peptide substrate, as analyzed by surface plasmon resonance, showed that alpha-MPP bound a peptide substrate as efficiently as MPP. The data suggest that the alpha-subunit is responsible for the binding of mitochondrial presequences prior their presentation to the catalytic site of MPP.
Luciano
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P
Laboratoire d'Ingéniérie des Systèmes Macromoléculaires, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, Marseille Cedex 20, 13402, France.
Geoffroy
S
S
Brandt
A
A
Hernandez
J F
JF
Géli
V
V
D000409:Alanine; D001665:Binding Sites; D015374:Biosensing Techniques; D004926:Escherichia coli; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000096122:Mitochondrial Processing Peptidase
D000409:Alanine; D008666:Metalloendopeptidases; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D000096122:Mitochondrial Processing Peptidase