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© 2015 John Wiley & Sons Ltd. The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer. We have identified amino acid substitutions in the Escherichia coli TatC protein that block the function of the wild type TatC when the two proteins are co-produced. We show that in the presence of a substrate protein two TatC wild type protomers come into close proximity, allowing a disulphide bond to form across position M205C. The amino acid variants abolish this substrate-induced disulphide crosslink. Our findings show that TatC functions as an obligate multimer.

Original publication

DOI

10.1111/mmi.13106

Type

Journal article

Journal

Molecular Microbiology

Publication Date

01/10/2015

Volume

98

Pages

111 - 129