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
- Authors
- Lee, AY; Park, SG; Kho, CW; Park, SY; Cho, S; Lee, SC; Lee, DH; Myung, PK; Park, BC
- Issue Date
- Nov-2004
- Publisher
- WILEY-V C H VERLAG GMBH
- Keywords
- degradomics; lsp-1; serine protease; two-dimensional gel
- Citation
- PROTEOMICS, v.4, no.11, pp 3437 - 3445
- Pages
- 9
- Journal Title
- PROTEOMICS
- Volume
- 4
- Number
- 11
- Start Page
- 3437
- End Page
- 3445
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/24744
- DOI
- 10.1002/pmic.200400997
- ISSN
- 1615-9853
1615-9861
- 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.
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