Abstract
The thylakoid lumen is a membrane-enclosed aqueous compartment. Growing evidence indicates that the thylakoid lumen is not only a sink for protons and inorganic ions translocated during photosynthetic reactions but also a place for metabolic activities, e.g. proteolysis of photodamaged proteins, to sustain efficient photosynthesis. However, the mechanism whereby organic molecules move across the thylakoid membranes to sustain these lumenal activities is not well understood. In a recent study of Cyanophora paradoxa chloroplasts (muroplasts), we fortuitously detected a conspicuous diffusion channel activity in the thylakoid membranes. Here, using proteoliposomes reconstituted with the thylakoid membranes from muroplasts and from two other phylogenetically distinct organisms, cyanobacterium Synechocystis sp. PCC 6803 and spinach, we demonstrated the existence of nonselective channels large enough for enabling permeation of small organic compounds (e.g. carbohydrates and amino acids with Mr < 1500) in the thylakoid membranes. Moreover, we purified, identified, and characterized a muroplast channel named here CpTPOR. Osmotic swelling experiments revealed that CpTPOR forms a nonselective pore with an estimated radius of ∼1.3 nm. A lipid bilayer experiment showed variable-conductance channel activity with a typical single-channel conductance of 1.8 nS in 1 m KCl with infrequent closing transitions. The CpTPOR amino acid sequence was moderately similar to that of a voltage-dependent anion-selective channel of the mitochondrial outer membrane, although CpTPOR exhibited no obvious selectivity for anions and no voltage-dependent gating. We propose that transmembrane diffusion pathways are ubiquitous in the thylakoid membranes, presumably enabling rapid transfer of various metabolites between the lumen and stroma.
Highlights
The thylakoid lumen is a membrane-enclosed aqueous compartment
To clarify whether the presence of organic molecule– permeable channels is a common characteristic of the thylakoid membrane, we separated the thylakoid membranes from the muroplasts, PCC 6803 cells, and S. oleracea chloroplasts by sucrose density gradient centrifugation and prepared proteoliposomes reconstituted with each membrane preparation
The permeability of these proteoliposomes was examined by a liposome-swelling assay [22] in which the swelling of the proteoliposomes caused by the influx of tested substrates is monitored by measuring the reduction of the optical density at 400 nm (A400) of the reaction mixture (Fig. 1)
Summary
Dye Lucifer yellow (Mr of 444.24), supporting the possible existence of pore-like channel(s). The molecular identity of the channel remained unknown, and the permeability of organic molecules across the thylakoid membranes has not been explored further. In this study, using proteoliposomes reconstituted with the thylakoid membrane proteins of muroplasts, cyanobacterium Synechocystis sp. PCC 6803 (referred to as PCC 6803) and spinach (Spinacia oleracea) chloroplasts, we demonstrated the presence of nonselective channels with a pore size large enough to allow permeation of organic molecules. We purified, identified, and characterized the muroplast channel by employing biochemical and electrophysiological approaches
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have