title: Identification of the degradome of Isp-1, a major intracellular serine protease of Bacillus subtilis, by two-dimensional gel electrophoresis and matrix- assisted laser desorption/ionization-time of flight analysis.
abstract: Intracellular serine protease-1 (Isp-1) is a major intracellular serine protease of Bacillus subtilis, whose functions still remain largely unknown. Furthermore, physiological substrates are yet to be determined. To identify Isp-1 substrates, we digested extract obtained from an Isp-1 deficient Bacillus mutant with purified Isp-1 and examined eliminated or decreased spots by two-dimensional gel and matrix-assisted laser desorption/ionization-time of flight analyses. Proteins degraded by Isp-1, termed the Isp-1 degradome, are involved in a variety of cellular functions such as DNA packing, genetic competence, and protein secretion. From the degradome we selected ClpC and EF-Tu as putative Isp-1 substrates and studied their in vitro degradation. ClpC and EF-Tu contain putative cleavage sites for Isp-1. N-terminal sequencing of in vitro proteolytic fragments of ClpC and EF-Tu revealed that these sites are indeed recognized and cleaved by Isp-1. Moreover, the cellular levels of ClpC and EF-Tu were dramatically reduced at the late stationary phase, where the expression level of Isp-1 was greatly increased. These results suggest that the regulated proteolysis of ClpC by Isp-1 plays an important role in the stationary phase adaptive response. This degradomic approach could provide a powerful tool for finding physiological substrates of many proteolytic enzymes whose functions remain to be determined.
authors.affiliation: Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea.
authors.forename: Ah Young
authors.initials: AY
authors.lastname: Lee
authors.forename: Sung
authors.initials: S
authors.lastname: Goo Park
authors.forename: Chang Won
authors.initials: CW
authors.lastname: Kho
authors.forename: Sun
authors.initials: S
authors.lastname: Young Park
authors.forename: Sayeon
authors.initials: S
authors.lastname: Cho
authors.forename: Sang Chul
authors.initials: SC
authors.lastname: Lee
authors.forename: Do Hee
authors.initials: DH
authors.lastname: Lee
authors.forename: Pyung Keun
authors.initials: PK
authors.lastname: Myung
authors.forename: Byoung Chul
authors.initials: BC
authors.lastname: Park
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D015180:Electrophoresis, Gel, Two-Dimensional; D012697:Serine Endopeptidases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D013997:Time Factors
chemical_list: D012697:Serine Endopeptidases