Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts.
We have recently isolated and identified a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli. We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate specificity and intracellular localization. Mutagenesis studies of the residues involved in metal binding identified the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate specificity studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se. Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro. In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria--degradation of targeting peptides.
Moberg
Per
P
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden.
Ståhl
Annelie
A
Bhushan
Shashi
S
Wright
Sarah J
SJ
Eriksson
AnnaCarin
A
Bruce
Barry D
BD
Glaser
Elzbieta
E
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D017124:Conserved Sequence; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism*
D029681:Arabidopsis Proteins; D010455:Peptides; D010940:Plant Proteins; D008666:Metalloendopeptidases; D015032:Zinc