title: Two novel targeting peptide degrading proteases, PrePs, in mitochondria and chloroplasts, so similar and still different.
abstract: Two novel metalloproteases from Arabidopsis thaliana, termed AtPrePI and AtPrePII, were recently identified and shown to degrade targeting peptides in mitochondria and chloroplasts using an ambiguous targeting peptide. AtPrePI and AtPrePII are classified as dually targeted proteins as they are targeted to both mitochondria and chloroplasts. Both proteases harbour an inverted metal binding motif and belong to the pitrilysin subfamily A. Here we have investigated the subsite specificity of AtPrePI and AtPrePII by studying their proteolytic activity against the mitochondrial F(1)beta pre-sequence, peptides derived from the F(1)beta pre-sequence as well as non-mitochondrial peptides and proteins. The degradation products were analysed, identified by MALDI-TOF spectrometry and superimposed on the 3D structure of the F(1)beta pre-sequence. AtPrePI and AtPrePII cleaved peptides that are in the range of 10 to 65 amino acid residues, whereas folded or longer unfolded peptides and small proteins were not degraded. Both proteases showed preference for basic amino acids in the P(1) position and small, uncharged amino acids or serine residues in the P'(1) position. Interestingly, both AtPrePI and AtPrePII cleaved almost exclusively towards the ends of the alpha-helical elements of the F(1)beta pre-sequence. However, AtPrePI showed a preference for the N-terminal amphiphilic alpha-helix and positively charged amino acid residues and degraded the F(1)beta pre-sequence into 10-16 amino acid fragments, whereas AtPrePII did not show any positional preference and degraded the F(1)beta pre-sequence into 10-23 amino acid fragments. In conclusion, despite the high sequence identity between AtPrePI and AtPrePII and similarities in cleavage specificities, cleavage site recognition differs for both proteases and is context and structure dependent.
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden. annelie_s@dbb.su.se
authors.forename: Annelie
authors.initials: A
authors.lastname: Ståhl
authors.forename: Stefan
authors.initials: S
authors.lastname: Nilsson
authors.forename: Pontus
authors.initials: P
authors.lastname: Lundberg
authors.forename: Shashi
authors.initials: S
authors.lastname: Bhushan
authors.forename: Henrik
authors.initials: H
authors.lastname: Biverståhl
authors.forename: Per
authors.initials: P
authors.lastname: Moberg
authors.forename: Magali
authors.initials: M
authors.lastname: Morisset
authors.forename: Alexander
authors.initials: A
authors.lastname: Vener
authors.forename: Lena
authors.initials: L
authors.lastname: Mäler
authors.forename: Ulo
authors.initials: U
authors.lastname: Langel
authors.forename: Elzbieta
authors.initials: E
authors.lastname: Glaser
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D002942:Circular Dichroism; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D010446:Peptide Fragments; D010447:Peptide Hydrolases; C501884:PrePI protein, Arabidopsis; C501885:PrePII protein, Arabidopsis