Author/Editor		Koronakis, Eva; Hughes, Colin; Milisav, Irina; Koronakis, Vassilis
Title		Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB
Type		članek
Source		Mol Microbiol
Vol. and No.		Letnik 16, št. 1
Publication year		1995
Volume		str. 87-96
Language		eng
Abstract		The Escherichia coli toxin exporter HIyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding casette (ABC) superfamily, and it directs translocation of the 110 kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HIyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the In vivo export ot protein by HIyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-bindlng fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HIyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vltro. The data show that protein export by HIyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokary- otic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.
Descriptors		ADENOSINETRIPHOSPHATASE ESCHERICHIA COLI ADENOSINE TRIPHOSPHATE HEMOLYSINS MUTATION MOLECULAR SEQUENCE DATA PROTEIN CONFORMATION MUTAGENESIS, SITE-DIRECTED KINETICS RECOMBINANT FUSION PROTEINS RESTRICTION MAPPING STRUCTURE-ACTIVITY RELATIONSHIP SERINE PROTEINASES