The protease-associated domain and C-terminal extension are required for zymogen processing, sorting within the secretory pathway, and activity of tomato subtilase 3 (SlSBT3).
ABSTRACT A transgenic plant cell suspension culture was established as a versatile and efficient expression system for the subtilase SlSBT3 from tomato. The recombinant protease was purified to homogeneity from culture supernatants by fractionated ammonium sulfate precipitation, batch adsorption to cation exchange material, and anion exchange chromatography. Purified SlSBT3 was identified as a 79-kDa glycoprotein with both complex and paucimannosidic type glycan chains at Asn(177), Asn(203), Asn(376), Asn(697), and Asn(745). SlSBT3 was found to be a very stable enzyme, being fully active at 60 degrees C and showing highest activity at alkaline conditions with a maximum between pH 7.5 and 8.0. Substrate specificity of SlSBT3 was analyzed in detail, revealing a preference for Gln and Lys in the P(1) and P(2) positions of oligopeptide substrates, respectively. Similar to bacterial, yeast, and mammalian subtilases, SlSBT3 is synthesized as a preproenzyme, and processing of the prodomain in the endoplasmic reticulum is a prerequisite for passage through the secretory pathway. SlSBT3 S538A and S538C active site mutants accumulated intracellularly as unprocessed zymogens, indicating that prodomain cleavage occurs autocatalytically. The wild-type SlSBT3 protein failed to cleave the prodomain of the S538A mutant in trans, demonstrating that zymogen maturation is an intramolecular process. Distinguishing features of plant as compared with mammalian subtilases include the insertion of a large protease-associated domain between the His and Ser residues of the catalytic triad and the C-terminal extension to the catalytic domain. Both features were found to be required for SlSBT3 activity and, consequently, for prodomain processing and secretion.
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ABSTRACT: The propeptide of furin has multiple roles in guiding the activation of the endoprotease in vivo. The 83-residue N-terminal propeptide is autoproteolytically excised in the endoplasmic reticulum (ER) at the consensus furin site, -Arg(104)-Thr-Lys-Arg(107)-, but remains bound to furin as a potent autoinhibitor. Furin lacking the propeptide is ER-retained and proteolytically inactive. Co-expression with the propeptide, however, restores trans-Golgi network (TGN) localization and enzyme activity, indicating that the furin propeptide is an intramolecular chaperone. Blocking this step results in localization to the ER-Golgi intermediate compartment (ERGIC)/cis-Golgi network (CGN), suggesting the ER and ERGIC/CGN recognize distinct furin folding intermediates. Following transport to the acidified TGN/endosomal compartments, furin cleaves the bound propeptide at a second, internal P1/P6 Arg site (-Arg-Gly-Val(72)-Thr-Lys-Arg(75)-) resulting in propeptide dissociation and enzyme activation. Cleavage at Arg(75), however, is not required for proper furin trafficking. Kinetic analyses of peptide substrates indicate that the sequential pH-modulated propeptide cleavages result from the differential recognition of these sites by furin. Altering this preference by converting the internal site to a canonical P1/P4 Arg motif (Val(72) --> Arg) caused ER retention and blocked activation of furin, demonstrating that the structure of the furin propeptide mediates folding of the enzyme and directs its pH-regulated, compartment-specific activation in vivo.Journal of Biological Chemistry 04/2002; 277(15):12879-90. · 4.65 Impact Factor
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ABSTRACT: Wild-type stomata are distributed nonrandomly, and their density is controlled by endogenous and exogenous factors. In the Arabidopsis mutant stomatal density and distribution1-1 (sdd1-1), the establishment of the stomatal pattern is disrupted, resulting in stomata clustering and twofold to fourfold increases in stomatal density. The SDD1 gene that encodes a subtilisin-like Ser protease is expressed strongly in stomatal precursor cells (meristemoids and guard mother cells), and the SDD1 promoter is controlled negatively by a feedback mechanism. The encoded protein is exported to the apoplast and probably is associated with the plasma membrane. SDD1 overexpression in the wild type leads to a phenotype opposite to that caused by the sdd1-1 mutation, with a twofold to threefold decrease in stomatal density and the formation of arrested stomata. While SDD1 overexpression was effective in the flp mutant, the tmm mutation acted epistatically. Thus, we propose that SDD1 generates an extracellular signal by meristemoids/guard mother cells and demonstrate that the function of SDD1 is dependent on TMM activity.The Plant Cell 08/2002; 14(7):1527-39. · 9.25 Impact Factor
- Biochemical and Biophysical Research Communications 05/1967; 27(2):157-62. · 2.41 Impact Factor