Fast Diffusion of the Unassembled PetC1-GFP Protein in the Cyanobacterial Thylakoid Membrane.

Radek Kaňa,Roman Sobotka,Josef Komenda,György Vámosi,Gábor Steinbach

doi:10.3390/life11010015

Abstract

Biological membranes were originally described as a fluid mosaic with uniform distribution of proteins and lipids. Later, heterogeneous membrane areas were found in many membrane systems including cyanobacterial thylakoids. In fact, cyanobacterial pigment–protein complexes (photosystems, phycobilisomes) form a heterogeneous mosaic of thylakoid membrane microdomains (MDs) restricting protein mobility. The trafficking of membrane proteins is one of the key factors for long-term survival under stress conditions, for instance during exposure to photoinhibitory light conditions. However, the mobility of unbound ‘free’ proteins in thylakoid membrane is poorly characterized. In this work, we assessed the maximal diffusional ability of a small, unbound thylakoid membrane protein by semi-single molecule FCS (fluorescence correlation spectroscopy) method in the cyanobacterium Synechocystis sp. PCC6803. We utilized a GFP-tagged variant of the cytochrome b6f subunit PetC1 (PetC1-GFP), which was not assembled in the b6f complex due to the presence of the tag. Subsequent FCS measurements have identified a very fast diffusion of the PetC1-GFP protein in the thylakoid membrane (D = 0.14 − 2.95 µm2s−1). This means that the mobility of PetC1-GFP was comparable with that of free lipids and was 50–500 times higher in comparison to the mobility of proteins (e.g., IsiA, LHCII—light-harvesting complexes of PSII) naturally associated with larger thylakoid membrane complexes like photosystems. Our results thus demonstrate the ability of free thylakoid-membrane proteins to move very fast, revealing the crucial role of protein–protein interactions in the mobility restrictions for large thylakoid protein complexes.

Highlights

Photochemical reactions in thylakoid membranes (TM) of oxygenic photoautotrophs are driven by light-induced charge separation within Photosystem II (PSII) and Photosystem I (PSI) complexes
We found that GFP-tagging of PetC1 inhibits its incorporation into the cyt b6 f complex and this fusion protein accumulates in TMs as a free protein (Figure 2)
Our fluorescence correlation spectroscopy (FCS) data showed that the unassembled TM proteins are able to diffuse very fast

Summary

Introduction

Photochemical reactions in thylakoid membranes (TM) of oxygenic photoautotrophs are driven by light-induced charge separation within Photosystem II (PSII) and Photosystem I (PSI) complexes. The primary photochemical reactions (light-harvesting, charge separation) require close nano-scale interactions of photosystems with the antenna proteins (e.g., cyanobacterial Phycobilisomes (PBS)) for the efficient flow of excitations [2]. The subsequent electron transport requires an optimal arrangement of all protein components forming the electron transport chain [3] In this respect, the association of the immobile protein complexes into nano-scale large supercomplexes (nanodomains) is one from the possible strategies in thylakoids [4,5]. Despite the small size of these nanodomains (up to 0.2 μm in diameter), which is close to the resolution limit of standard microscopes, some of them can be visualized either in isolated thylakoids by AFM microscopy [5] while others by confocal microscopy in native cells (see, e.g., [6,7,8]). PCC6803 (hereafter referred to as Synechocystis) (see, e.g., [9])

Methods

Discussion

Conclusion