title: Structural properties of substrate proteins determine their proteolysis by the mitochondrial AAA+ protease Pim1.
abstract: The protease Pim1/LON, a member of the AAA+ family of homo-oligomeric ATP-dependent proteases, is responsible for the degradation of soluble proteins in the mitochondrial matrix. To establish the molecular parameters required for the specific recognition and proteolysis of substrate proteins by Pim1, we analyzed the in organello degradation of imported reporter proteins containing different structural properties. The amino acid composition at the amino-terminal end had no major effect on the proteolysis reaction. However, proteins with an amino-terminal extension of less than 60 amino acids in front of a stably folded reporter domain were completely resistant to proteolysis by Pim1. Substrate proteins with a longer amino-terminal extension showed incomplete proteolysis, resulting in the generation of a defined degradation fragment. We conclude that Pim1-mediated protein degradation is processive and is initiated from an unstructured amino-terminal segment. Resistance to degradation and fragment formation was abolished if the folding state of the reporter domain was destabilized, indicating that Pim1 is not able to unravel folded proteins for proteolysis. We propose that the requirement for an exposed, large, non-native protein segment, in combination with a limited unfolding capability, accounts for the selectivity of the protease Pim1 for damaged or misfolded polypeptides.
authors.affiliation: Institut für Biochemie und Molekularbiologie, Universität Freiburg, Hermann-Herder-Str. 7, D-79104 Freiburg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: von Janowsky
authors.forename: Karin
authors.initials: K
authors.lastname: Knapp
authors.forename: Tamara
authors.initials: T
authors.lastname: Major
authors.forename: Martin
authors.initials: M
authors.lastname: Krayl
authors.forename: Bernard
authors.initials: B
authors.lastname: Guiard
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Voos
mesh_terms: D049069:ATP-Dependent Proteases / Q000737:chemistry / Q000378:metabolism*; D015153:Blotting, Western; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D017930:Genes, Reporter; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D024101:Mitochondrial Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D042844:L-Lactate Dehydrogenase (Cytochrome); D010450:Endopeptidases; D049069:ATP-Dependent Proteases; C085155:PIM1 protein, S cerevisiae; D012697:Serine Endopeptidases