title: Functional Characterization of Propeptides in Plant Subtilases as Intramolecular Chaperones and Inhibitors of the Mature Protease.
abstract: Subtilisin-like serine proteases (SBTs) are extracellular proteases that depend on their propeptides for zymogen maturation and activation. The function of propeptides in plant SBTs is poorly understood and was analyzed here for the propeptide of tomato subtilase 3 (SBT3PP). SBT3PP was found to be required as an intramolecular chaperone for zymogen maturation and secretion of SBT3 in vivo Secretion was impaired in a propeptide-deletion mutant but could be restored by co-expression of the propeptide in trans SBT3 was inhibited by SBT3PP with a Kd of 74 nm for the enzyme-inhibitor complex. With a melting point of 87 °C, thermal stability of the complex was substantially increased as compared with the free protease suggesting that propeptide binding stabilizes the structure of SBT3. Even closely related propeptides from other plant SBTs could not substitute for SBT3PP as a folding assistant or autoinhibitor, revealing high specificity for the SBT3-SBT3PP interaction. Separation of the chaperone and inhibitor functions of SBT3PP in a domain-swap experiment indicated that they are mediated by different regions of the propeptide and, hence, different modes of interaction with SBT3. Release of active SBT3 from the autoinhibited complex relied on a pH-dependent cleavage of the propeptide at Asn-38 and Asp-54. The remarkable stability of the autoinhibited complex and pH dependence of the secondary cleavage provide means for stringent control of SBT3 activity, to ensure that the active enzyme is not released before it reaches the acidic environment of the trans-Golgi network or its final destination in the cell wall.
authors.affiliation: From the Institute of Plant Physiology and Biotechnology and.
authors.forename: Michael
authors.initials: M
authors.lastname: Meyer
authors.affiliation: the Department of Microbiology, University of Hohenheim, D-70593 Stuttgart, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Leptihn
authors.affiliation: From the Institute of Plant Physiology and Biotechnology and.
authors.forename: Max
authors.initials: M
authors.lastname: Welz
authors.affiliation: From the Institute of Plant Physiology and Biotechnology and andreas.schaller@uni-hohenheim.de.
authors.forename: Andreas
authors.identifier: 0000-0001-6872-9576
authors.initials: A
authors.lastname: Schaller
mesh_terms: D002473:Cell Wall / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics; D008954:Models, Biological; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D059828:Plant Cells / Q000201:enzymology; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000201:enzymology / Q000235:genetics
chemical_list: D004792:Enzyme Precursors; D018832:Molecular Chaperones; D013381:Subtilisins; C119297:subtilase, plant
keywords: intramolecular chaperone; pH regulation; prodomain; protease inhibitor; protein folding; protein processing; serine protease; subtilase; zymogen