Protein exporter function and in vitro ATPase activity are correlated in ABC-domain mutants of HlyB.
The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB 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 vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.
Koronakis
E
E
Department of Pathology, Cambridge University, UK.
Hughes
C
C
Milisav
I
I
Koronakis
V
V
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000595:Amino Acid Sequence; D019286:Endopeptidase K; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000378:metabolism; D013329:Structure-Activity Relationship
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D018528:ATP-Binding Cassette Transporters; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D000251:Adenosine Triphosphatases; C086257:hemolysin B