Abstract
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.
Highlights
Substitution of glutamate 10 with alanine, glutamine, and even aspartate largely eliminated the ability of Tha[4] to complement transport, whereas a conservative substitution elsewhere in the transmembrane domain was without effect
Recent experiments have demonstrated that physiological quantities of Tha[4] can be integrated into isolated thylakoids (10). These data suggested the possibility that exogenous Tha[4] could complement a Tha[4] deficiency or Tha[4] inactivated with antibodies. i.e. that transport capability to ␣-Tha4-treated membranes could be restored by integration of exogenous Tha[4]
Because exogenous Tha[4] can interact with the Tha[4] antibody, it was important to rule out competitive release of the antibody, which would free endogenous Tha[4] to participate in the transport reaction
Summary
Twin arginine transport; PI, preimmune; IB, import buffer; DT17 or DT23, precursor form of the 17- or 23 kDa subunits of the oxygen-evolving complex of photosystem II containing a modified, truncated transit peptide; mOE17 or mOE23, mature form of the 17- or 23-kDa subunits of the oxygen evolving complex of photosystem II, respectively; Tricine, N-[2-hydroxy1,1-bis(hydroxymethyl)ethyl]glycine. The Tat system appears to employ, minimally, three integral membrane protein components, Tha[4], Hcf[106], and cpTatC, in thylakoids and TatA, TatB, and TatC, respectively, in bacteria (3, 4). Complementation of Tha[4] in Thylakoid Tat Translocation tamate (Glu) motif is conserved among all Hcf[106] and Tha[4] proteins, as is a glycine (Gly)-Pro motif between the transmembrane helix and the amphipathic helix (3). Despite this similarity, Tha[4] and Hcf[106] appear to perform very different functions. In addition to providing a rapid assay for substituting endogenous Tha[4] with engineered Tha[4], our results provide insights into the molecular basis for ⌬pH-induced assembly of the thylakoid Tat translocase
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