Mutational analysis of CvaA in the highly conserved domain of the membrane fusion protein family.
The antibacterial peptide toxin colicin V (ColV) uses a dedicated signal sequence-independent export system for its secretion in Escherichia coli that involves the products of three genes, cvaA, cvaB, and tolC in this process. As a member of the membrane fusion protein (MFP) family, the CvaA protein has been proposed to interact with an outer membrane protein TolC via its C-terminal hydrophobic domain. The importance of this domain, which is highly conserved throughout the members of MFP family, was analyzed by use of site-directed mutagenesis of missense or nonsense mutations with suppressors. All the nonsense mutations tested resulted in the loss of ColV secretion, indicating the importance of the C-terminus of CvaA, including the last 100 residue-hydrophilic domain. The missense mutations of several conserved amino acids have no drastic effects. On the other hand, when Glu-248, Ala-262, Thr-274, Leu-285, Gly-313, Ala-322, or Val-335 of CvaA protein was mutated, the secretion of ColV was greatly reduced in certain mutants. While some mutations resulted in structural instability, Glu-248 to Lys and Ala-322 to Gly proteins were relatively stable, but were not functional in ColV secretion. The results indicate that these conserved amino acids are important for the structure and functions of CvaA in the secretion of ColV.
Hwang
J
J
Department of Biology, 24 Peachtree Center Ave., 402 Kell Hall, Georgia State University, Atlanta, GA 3030, USA.
Tai
P C
PC
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D015153:Blotting, Western; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods
D018528:ATP-Binding Cassette Transporters; C096447:CvaA protein, E coli; D029968:Escherichia coli Proteins; D008565:Membrane Proteins