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.
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.
Lee
Ah Young
AY
Systemic Proteomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, South Korea.
Goo Park
Sung
S
Kho
Chang Won
CW
Young Park
Sun
S
Cho
Sayeon
S
Lee
Sang Chul
SC
Lee
Do Hee
DH
Myung
Pyung Keun
PK
Park
Byoung Chul
BC
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
D012697:Serine Endopeptidases