ALB3 Insertase Mediates Cytochrome b6 Co-translational Import into the Thylakoid Membrane.

Jarosław Króliczewski,Bożena Króliczewska,Małgorzata Piskozub,Rafał Bartoszewski

doi:10.1038/srep34557

Abstract

The cytochrome b6 f complex occupies an electrochemically central position in the electron-transport chain bridging the photosynthetic reaction center of PS I and PS II. In plants, the subunits of these thylakoid membrane protein complexes are both chloroplast and nuclear encoded. How the chloroplast-encoded subunits of multi-spanning cytochrome b6 are targeted and inserted into the thylakoid membrane is not fully understood. Experimental approaches to evaluate the cytochrome b6 import mechanism in vivo have been limited to bacterial membranes and were not a part of the chloroplast environment. To evaluate the mechanism governing cytochrome b6 integration in vivo, we performed a comparative analysis of both native and synthetic cytochrome b6 insertion into purified thylakoids. Using biophysical and biochemical methods, we show that cytochrome b6 insertion into the thylakoid membrane is a non-spontaneous co-translational process that involves ALB3 insertase. Furthermore, we provided evidence that CSP41 (chloroplast stem–loop-binding protein of 41 kDa) interacts with RNC-cytochrome b6 complexes, and may be involved in cytochrome b6 (petB) transcript stabilization or processing.

Highlights

We provided evidence that CSP41 interacts with ribosome-nascent chain complexes (RNCs)-cytochrome b6 complexes, and may be involved in cytochrome b6 transcript stabilization or processing
The majority of proteins incorporated into the thylakoid membrane utilize the spontaneous or signal recognition particle (SRP) pathway, while protein transport through the thylakoid membrane is mediated by the Sec or ΔpH/Tat pathway[6]
The circular dichroism (CD) spectra of isolated native cytochrome b6 indicated a high proportion of the α-helical structure characterised by negative maxima at 208 and 222 nm (Fig. 1A and Table 1)

Summary

Introduction

We provided evidence that CSP41 (chloroplast stem–loop-binding protein of 41 kDa) interacts with RNC-cytochrome b6 complexes, and may be involved in cytochrome b6 (petB) transcript stabilization or processing. Cytochrome b6 f and PS I complex participate in cyclic electron transfer, which generates an electrochemical proton gradient across the thylakoid membrane without net production of reducing equivalents[3]. For the import into the thylakoid membrane of proteins from both nuclear and chloroplast origin, four independent precursor-specific transport pathways had been proposed (classified on the basis of their energy and stromal factor requirements)[6]. These four pathways have been categorized as “spontaneous”, signal recognition particle (SRP), secretory (Sec), and twin-arginine translocase-dependent (ΔpH/Tat)[7]. The spontaneous mechanism was shown to be active for some of nuclear-encoded multi-spanning thylakoid membrane proteins including the PsaK and PsaG subunits of PS I11,12

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