Isolation and identification of a novel mitochondrial metalloprotease (PreP) that degrades targeting presequences in plants.
Most of the nuclear encoded mitochondrial precursor proteins contain an N-terminal extension called the presequence that carries targeting information and that is cleaved off after import into mitochondria. The presequences are amphiphilic, positively charged, membrane-interacting peptides with a propensity to form alpha-helices. Here we have investigated the proteolysis of the presequences that have been cleaved off inside mitochondria. A presequence derived from the overexpressed F(1)beta subunit of the ATP synthase and specific synthetic fluorescent peptides (Pep Tag Protease assay) have been shown to undergo rapid degradation catalyzed by a matrix located protease. We have developed a three-step chromatographic procedure including affinity and anion exchange chromatography for isolation of the protease from potato tuber mitochondria. Two-dimensional gel electrophoresis of the isolated proteolytically active fraction followed by electrospray ionization-mass spectrometry/mass spectrometry and data base searches allowed identification of the presequence peptide-degrading protease in Arabidopsis thaliana data base as a novel mitochondrial metalloendoprotease with a molecular mass of 105 kDa. The identified metalloprotease contains an inverted zinc-binding motif and belongs to the pitrilysin family.
Stahl
Annelie
A
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
Moberg
Per
P
Ytterberg
Jimmy
J
Panfilov
Oleg
O
Brockenhuus Von Lowenhielm
Helena
H
Nilsson
Fredrik
F
Glaser
Elzbieta
E
D000595:Amino Acid Sequence; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D011498:Protein Precursors / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism*
D024101:Mitochondrial Proteins; D010940:Plant Proteins; D011498:Protein Precursors; D008666:Metalloendopeptidases; D006180:Proton-Translocating ATPases