Functional complexity of the twin-arginine translocase TatC component revealed by site-directed mutagenesis.
The Escherichia coli Tat apparatus is a membrane-bound protein translocase that serves to export folded proteins synthesized with N-terminal twin-arginine signal peptides. The essential TatC component of the Tat translocase is an integral membrane protein probably containing six transmembrane helices. Sequence analysis identified conserved TatC amino acid residues, and the role of these side-chains was assessed by single alanine substitution. This approach identified three classes of TatC mutants. Class I mutants included F94A, E103A and D211A, which were completely devoid of Tat-dependent protein export activity and thus represented residues essential for TatC function. Cross-complementation experiments with class I mutants showed that co-expression of D211A with either F94A or E103A regenerated an active Tat apparatus. These data suggest that different class I mutants may be blocked at different steps in protein transport and point to the co-existence of at least two TatC molecules within each Tat translocon. Class II mutations identified residues important, but not essential, for Tat activity, the most severely affected being L99A and Y126A. Class III mutants showed no significant defects in protein export. All but three of the essential and important residues are predicted to cluster around the cytoplasmic N-tail and first cytoplasmic loop regions of the TatC protein.
Buchanan
Grant
G
Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, UK.
de Leeuw
Erik
E
Stanley
Nicola R
NR
Wexler
Margaret
M
Berks
Ben C
BC
Sargent
Frank
F
Palmer
Tracy
T
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021381:Protein Transport
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C461379:TatC protein, E coli; C408585:twin-arginine translocase complex, E coli