title: Catalysis, subcellular localization, expression and evolution of the targeting peptides degrading protease, AtPreP2.
abstract: We have previously identified a zinc metalloprotease involved in the degradation of mitochondrial and chloroplast targeting peptides, the presequence protease (PreP). In the Arabidopsis thaliana genomic database, there are two genes that correspond to the protease, the zinc metalloprotease (AAL90904) and the putative zinc metalloprotease (AAG13049). We have named the corresponding proteins AtPreP1 and AtPreP2, respectively. AtPreP1 and AtPreP2 show significant differences in their targeting peptides and the proteins are predicted to be localized in different compartments. AtPreP1 was shown to degrade both mitochondrial and chloroplast targeting peptides and to be dual targeted to both organelles using an ambiguous targeting peptide. Here, we have overexpressed, purified and characterized proteolytic and targeting properties of AtPreP2. AtPreP2 exhibits different proteolytic subsite specificity from AtPreP1 when used for degradation of organellar targeting peptides and their mutants. Interestingly, AtPreP2 precursor protein was also found to be dual targeted to both mitochondria and chloroplasts in a single and dual in vitro import system. Furthermore, targeting peptide of the AtPreP2 dually targeted green fluorescent protein (GFP) to both mitochondria and chloroplasts in tobacco protoplasts and leaves using an in vivo transient expression system. The targeting of both AtPreP1 and AtPreP2 proteases to chloroplasts in A. thaliana in vivo was confirmed via a shotgun mass spectrometric analysis of highly purified chloroplasts. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that AtPreP1 and AtPreP2 are differentially expressed in mature A. thaliana plants. Phylogenetic evidence indicated that AtPreP1 and AtPreP2 are recent gene duplicates that may have diverged through subfunctionalization.
authors.affiliation: Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden.
authors.forename: Shashi
authors.initials: S
authors.lastname: Bhushan
authors.forename: Annelie
authors.initials: A
authors.lastname: Ståhl
authors.forename: Stefan
authors.initials: S
authors.lastname: Nilsson
authors.forename: Benoit
authors.initials: B
authors.lastname: Lefebvre
authors.forename: Motoaki
authors.initials: M
authors.lastname: Seki
authors.forename: Christian
authors.initials: C
authors.lastname: Roth
authors.forename: David
authors.initials: D
authors.lastname: McWilliam
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Wright
authors.forename: David A
authors.initials: DA
authors.lastname: Liberles
authors.forename: Kazuo
authors.initials: K
authors.lastname: Shinozaki
authors.forename: Barry D
authors.initials: BD
authors.lastname: Bruce
authors.forename: Marc
authors.initials: M
authors.lastname: Boutry
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D001693:Biological Transport, Active; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression; D017343:Genes, Plant; D013058:Mass Spectrometry; D045726:Metalloproteases / Q000235:genetics* / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D030821:Plants, Genetically Modified; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
chemical_list: D018076:DNA, Complementary; D018744:DNA, Plant; D010455:Peptides; D045726:Metalloproteases