Requirement of a Tha4-conserved transmembrane glutamate in thylakoid Tat translocase assembly revealed by biochemical complementation.
The thylakoid Tat system employs three membrane components and the pH gradient to transport folded proteins. The translocase is signal-assembled, i.e. a receptor complex containing cpTatC and Hcf106 binds the precursor protein, and upon membrane energization, Tha4 is recruited to the precursor-receptor complex to effect translocation. We developed a two-step complementation assay to examine the implied central role of Tha4 in translocation. The first step results in the inactivation of endogenous Tha4 with specific antibodies. The second step involves integrating exogenous Tha4 and presenting the system with precursor protein. We verified this approach by confirming the results obtained recently with the Escherichia coli Tha4 ortholog TatA, i.e. that the carboxyl terminus is dispensable and the amphipathic helix essential for transport. We then investigated a conserved Tha4 transmembrane glutamate in detail. Substitution of glutamate 10 with alanine, glutamine, and even aspartate largely eliminated the ability of Tha4 to complement transport, whereas a conservative substitution elsewhere in the transmembrane domain was without effect. Chemical cross-linking assays showed that the mutated Tha4s failed to be recruited to the receptor complex under transport conditions, indicating a role for the transmembrane glutamate in translocase assembly. This assay promises an avenue into understanding the role of Tha4 in both the assembly and translocation steps of the Tat translocase.
Dabney-Smith
Carole
C
Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, Florida 32611, USA.
Mori
Hiroki
H
Cline
Kenneth
K
D000409:Alanine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D005973:Glutamine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008957:Models, Genetic; D018532:Pisum sativum; D010940:Plant Proteins; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D020524:Thylakoids / Q000378:metabolism*
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D008565:Membrane Proteins; D010940:Plant Proteins; C120444:Tha4 protein, plant; D005973:Glutamine; D001224:Aspartic Acid; D018698:Glutamic Acid; D000409:Alanine