Functional reconstitution of TatB into the 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.

Dual targeting of TatA points to a chloroplast-like Tat pathway in plant mitochondria

“…His-tagged precursor proteins of mtRieske from potato and GrpE from Arabidopsis thaliana were obtained by overexpression in E. coli strain BL21(DE3) using the T7-based system developed by Studier and Moffat (1986) following the protocol of Zinecker et al (2020) with the following modifications: (i) cultures were grown in LB media supplemented with 0.4% glucose, 50 mg/ml kanamycin for 1.5 - 3 h after induction with IPTG, (ii) the His-tagged proteins were recovered from inclusion bodies solubilized in GuaHCl binding buffer (20 mM Hepes, 500 mM NaCl, 20 mM Imidazole, 6 M GuanidinHCl, pH 7.5), (iii) during Ni 2+ -affinity chromatography the column (HiTrap Chelating HP columns, GE Healthcare) was washed with (20 mM Hepes, 500 mM NaCl, 20 mM Imidazole, 8 M urea, pH 7.5) and eluted with (20 mM Hepes, 500 mM NaCl, 500 mM Imidazole, 8 M urea, pH 7.5), (iv) the samples were concentrated using Vivaspin 3.000 MWCO PES ultrafiltration columns (Sartorius AG, Göttingen, Germany) and finally dialysed against (10 mM Hepes, 5 mM MgCl 2 , 7 M urea, pH 8.0).…”

Expression of acid cleavable Asp-Pro linked multimeric AFP peptide in E. coli

“…Denaturing conditions can be detrimental to enzyme activity, while most peptide multimers tend to form aggregates. Alternatively, chemical cleavage has lower reagent costs, wider temperature and suitable pH range, solubilization agent compatibility, and shorter artificial sequence insertions [ 10 , 11 ]. Some peptide bonds are known to be less acid stable than others.…”