PMID:11100827

Citation	Yang, FL and Braun, V (2000) ShlB mutants of Serratia marcescens allow uncoupling of activation and secretion of the ShlA hemolysin. Int. J. Med. Microbiol. 290:529-38
Abstract	The ShlB protein in the outer membrane of Serratia marcescens secretes hemolytic ShlA protein into the culture medium. In the absence of ShlB, nonhemolytic ShlA remains in the periplasm. ShlB mutants were isolated in which secretion was uncoupled from activation. Mutants with a tetrapeptide insertion after residues 136 or 224 of mature ShlB and a mutant with an insertion after residue 154 and a deletion secreted inactive ShlA. In vitro, secreted nonhemolytic ShlA was converted into hemolytic ShlA by isolated wild-type ShlB and by complementation with an N-terminal ShlA fragment of 255 residues (ShlA-255). The isolation of secretion-competent, but activation-negative mutants indicates that secretion alone is not sufficient for activation of ShlA. Rather, ShlB is required for activation and secretion, and the mutants define sites in ShlB which are involved in activation. According to a predicted transmembrane model of ShlB, the mutations that retain secretion competence but abolish activation competence are located in the most prominent surface loop and the following transmembrane loop. In one tetrapeptide insertion mutant, ShlB-332, most of the ShlA remained cell-associated in an inactive form and low amounts (6%) were hemolytic. Secreted inactive ShlA(o) was completely degraded by trypsin, in contrast to hemolytic ShlA, which was cleaved into two fragments of 60 and 100 kDa. This result indicates that the conformational change from a highly trypsin-sensitive to a highly trypsin-resistant protein with only a single cleavage site in a polypeptide of 1,578 residues occurs upon activation of ShlA and not during secretion.
Links	PubMed Online version:10.1016/S1438-4221(00)80018-1
Keywords	Alkaline Phosphatase/secretion; Bacterial Proteins/physiology; Hemolysin Proteins/chemistry; Hemolysin Proteins/metabolism; Membrane Proteins; Mutation; Protein Conformation; Serratia marcescens/physiology; Trypsin/pharmacology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
SERMA:HLYB		GO:0008320: protein transmembrane transporter activity	ECO:0000315:		F		Figure 1 shows that organisms lacking ShlB had no hemolytic activity since no ShlA was secreted. Mutants with small changes to ShlB were still able to secrete active ShlA.	complete CACAO 10057
SERMA:HLYB	enables	GO:0008320: protein transmembrane transporter activity	ECO:0000315: mutant phenotype evidence used in manual assertion		F		Seeded From UniProt	complete

References

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