Functional reconstitution of TatB into thylakoidal Tat translocase

Abstract

We have established an experimental system for the functional analysis of thylakoidal TatB, a component of the membrane-integral TatBC receptor complex of the thylakoidal Twinarginine protein transport (Tat 1 ) machinery. For this purpose, the intrinsic TatB activity of isolated pea thylakoids was inhibited by affinity-purified antibodies and substituted by supplementing the assays with TatB protein either obtained by in vitro translation or purified after heterologous expression in E. coli. Tat transport activity of such reconstituted thylakoids, which was analyzed with the authentic Tat substrate pOEC16, reached routinely 20 -25% of the activity of mock-treated thylakoid vesicles analysed in parallel. In contrast, supplementation of the assays with the purified antigen comprising all but the N-terminal transmembrane helix of thylakoidal TatB did not result in Tat transport reconstitution which confirms that transport relies strictly on the activity of the TatB protein added and is not due to restoration of the intrinsic TatB activity by antibody release. Unexpectedly, even a mutant TatB protein (TatB,E10C) assumed to be incapable of assembling into the TatBC receptor complex showed low but considerable transport reconstitution underlining the sensitivity of the approach and its suitability for further functional mutant analyses. Finally, quantification of TatB demand suggests that TatA and TatB are required in approximately equimolar amounts to achieve Tat-dependent thylakoid transport. Keywords Twin-arginine translocation, thylakoid membrane, transport reconstitution, heterologous overexpression, protein purification 1 The abbreviations used are: Tat, twin-arginine translocation; OEC16, 16 kDa subunit of the oxygen-evolving system associated with photosystem II; RTS, Rapid Translation System; Hepes, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; BrCN, cyanogen bromide -3 -

Functional reconstitution of TatB into the thylakoidal Tat translocase

“…Three models of TatA activity have been proposed, namely (i) TatA constituting an adaptable translocation pore that is temporarily formed on demand (Mori and Cline. 2002;Gohlke et al, 2005;Cline and McCaffery, 2007), (ii) local weakening of the membrane at the sites of TatA accumulation which allows protein translocation directly through the lipid bilayer (Natale et al, 2008;Hou et al, 2018), or (iii) TatA providing the (co-)enzymatic activity converting the entire Tat apparatus into the active translocase (Jakob et al, 2009;Hauer et al, 2013;. The latter is supported by the observation that TatA is not strictly associated with the thylakoid membrane but found in active form also soluble in the chloroplast stroma (Frielingsdorf et al, 2008).…”