Cleavage site selection within a folded substrate by the ATP-dependent lon protease.
Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.
Ondrovicová
Gabriela
G
Institute of Molecular Biology, Slovak Academy of Sciences, 84551 Bratislava, Slovak Republic.
Liu
Tong
T
Singh
Kamalendra
K
Tian
Bin
B
Li
Hong
H
Gakh
Oleksandr
O
Perecko
Dusan
D
Janata
Jirí
J
Granot
Zvi
Z
Orly
Joseph
J
Kutejová
Eva
E
Suzuki
Carolyn K
CK
D000255:Adenosine Triphosphate / Q000737:chemistry*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry; D049070:Protease La / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D000071636:Protein Phosphatase 2C; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors; D000098682:Steroidogenic Acute Regulatory Protein
D005656:Fungal Proteins; D010455:Peptides; D010750:Phosphoproteins; D011506:Proteins; D000098682:Steroidogenic Acute Regulatory Protein; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D049070:Protease La