Molecular Properties of Oep21, an ATP-regulated Anion-selective Solute Channel from the Outer Chloroplast Membrane

Abstract

The flux of phosphorylated carbohydrates, the major export products of chloroplasts, is regulated at the level of the inner and presumably also at the level of the outer membrane. This is achieved through modulation of the outer membrane Oep21 channel currents and tuning of its ion selectivity. Refined analysis of the Oep21 channel properties by biochemical and electrophysiological methods revealed a channel formed by eight ␤-strands with a wider pore vestibule of d vest ϳ2.4 nm at the intermembrane site and a narrower filter pore of d restr ϳ1 nm. The Oep21 pore contains two high affinity sites for ATP, one located at a relative transmembrane electrical distance ␦ ‫؍‬ 0.56 and the second close to the vestibule at the intermembrane site. The ATP-dependent current block and reduction in anion selectivity of the Oep21 channel is relieved by the competitive binding of phosphorylated metabolic intermediates like 3-phosphoglycerate and glycerinaldehyde 3-phosphate. Deletion of a C-terminal putative FX 4 K binding motif in Oep21 decreased the capability of the channel to tune its ion selectivity by about 50%, whereas current block remained unchanged.Plastid organelles perform vital biosynthetic functions in every plant organ. They are surrounded by double membranes, the inner and the outer envelope, which delimit spatially and temporally the plastid compartment from the cytoplasm. Both envelope membranes are distinguishable by their structure, function, and biochemical properties but also cooperate, for example, in the synthesis of lipids or in protein translocation. Chloroplasts are the site of carbon dioxide reduction and its assimilation into carbohydrates, amino acids, fatty acids, and terpenoid compounds. The manifold biosynthetic functions of chloroplasts require the existence of different selective transport mechanisms across the envelope membranes to provide the cell with carbohydrates, organic nitrogen, and sulfur compounds (1, 2 Until recently, the outer envelope membrane was considered to be freely permeable for most small molecular mass solutes up to 10 kDa (1).Correspondingly, it was believed that the osmotic barrier against the cytosol is formed exclusively by the inner envelope membrane, containing specific carrier proteins, some of which have been identified on the functional and also on the molecular level (2). However, our recent reports reveal the presence of several specific solute pores in the outer envelope, indicating that the intermembrane space is not freely accessible to low molecular weight solutes (3-6).Whereas the inner envelope carrier proteins (e.g. the triose phosphate-phosphate translocator, the dicarboxylic acid translocator, and the hexose phosphate carrier) show a distinct substrate selectivity and specificity, it remains elusive to what extent the transport through these outer membrane channels is regulated (7). The ancestral relation between plastids and Gram-negative bacteria suggests the presence of different solute channel proteins in the organellar outer membrane. In pea chlor...

AT_CHLORO, a Comprehensive Chloroplast Proteome Database with Subplastidial Localization and Curated Information on Envelope Proteins

“…These proteins could be classified into four categories. The first category included a series of five proteins (OEP21, OEP24, OEP37, and isoforms), one (OEP21-like) identified for the first time, one identified previously (73), and two more identified recently (74,75), were demonstrated to act as solute or ion channels. The second category included six members of the OEP16 protein family with four of them being previously known envelope proteins (26, 58).…”

OEP37 Is a New Member of the Chloroplast Outer Membrane Ion Channels

“…OEP16 shows similarities to components of the protein translocase of the inner mitochondrial membrane and to a lesser extent to LivH, an amino acid transporter in E. coli (12). In contrast, OEP21 has been shown to form an anion channel, which is regulated by ATP and triose phosphates from the intramembrane space (7,13). The channel properties of OEP24, however, closely resemble those described for general diffusion pores (3).…”

Chloroplast β-Barrel Proteins Are Assembled into the Mitochondrial Outer Membrane in a Process That Depends on the TOM and TOB Complexes

“…The constructs for the self-assembly GFP assays were amplified from A. thaliana cDNA using standard PCR techniques and subsequently cloned into the pAVA plasmid (25) containing the fragments for saGFP11 (N-/C-terminal) or saGFP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Templates for the saGFP(1-10) and saGFP11 fragments were obtained from Dr. G. S. Waldo (Los Alamos, NM).…”

“…The number of characterized ␤-barrel proteins in chloroplast OM is not much higher, although sequence analysis predicted the presence of many such proteins in this membrane (1). Some of those (for example, outer envelope proteins OEP21, OEP24, and OEP37) were proposed to function as pores for small metabolites, and their distinct substrate specificities may point to discrete roles in various metabolic processes (2,3). The chloroplast OM harbors also Toc75, a ␤-barrel protein with several isoforms in Arabidopsis thaliana (4).…”

“…In addition to the outer membrane (OM) 3 of Gram-negative bacteria, membrane-embedded ␤-barrel proteins are found also in the OM of the endosymbiotic organelles, mitochondria and chloroplasts. Compared with the diversity of ␤-barrel membrane proteins in prokaryotes, the number of organellar OM proteins confirmed to have this structural type is rather limited.…”