Characterization of the Free and Membrane-Associated Fractions of the Thylakoid Lumen Proteome in Arabidopsis thaliana.

Peter J Gollan,Azfar A Bajwa,Ilaria Mancini,Eva-Mari Aro,Andrea Trotta

doi:10.3390/ijms22158126

Abstract

The thylakoid lumen houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyze the contents of each proteome. These results identified 60 lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several protein oligomers in the thylakoid lumen.

Highlights

The thylakoid lumen proteome of Arabidopsis was obtained in two distinct fractions; the fraction of the proteome containing free, soluble proteins was collected as the supernatant after centrifugation of ruptured purified thylakoids, according to published lumen isolation methods [15,28]
We separated the thylakoid lumen proteome into two fractions; the soluble, free lumen (FL) fraction that was released upon inversion of thylakoid membranes [15,28], and the membrane-associated lumen (MAL) fraction that was dissociated from inverted thylakoids by interfering with both the electrostatic protein-protein interactions, using NaCl, and with the hydrogen bonding network, using the chaotropic agent urea [26,27]
The thylakoid lumen is vital for the assembly, function and maintenance of efficient photosynthetic electron transport, and several lumen proteins are known to be involved in these processes, but the functions of most lumen proteins remains elusive

Summary

Introduction

A folded and continuous network of thylakoid membranes physically separates the soluble stroma (exterior) and lumen (interior) compartments. The membranes are embedded with large protein complexes that use light energy to oxidize water molecules in the lumen and transport electrons, generating reducing equivalents in the stroma. Electron transport along the thylakoid membrane develops an electrochemical gradient across the membrane that is harnessed by ATP synthase to produce ATP. Reducing equivalents and ATP power the reduction of CO2 in the chloroplast stroma into the stored chemical energy of carbohydrates. In this process, which is known as photosynthesis, the thylakoid lumen holds water for oxidation by photosystem (PS)

Methods

Results

Discussion

Conclusion