| Regional Biophysics Meeting 2005, March 16-20, Zreče, Slovenia | [MembBiophys] |
Putative ion channels with probable chloroplast localization have been identified by bioinformatics in Arabidopsis genome and in the genome of cyanobacteria, which are considered to represent the precursors of chloroplasts. The translated protein of a gene encoding a putative potassium channel of Synechocystis PCC6803 (SynK) displays six transmembrane segments and a pore region between the fifth and sixth segments showing the typical potassium selectivity filter sequence. Three-dimensional modeling reveals a structure compatible with a shaker-like channel. The only prokaryotic shaker-like potassium channel known by far is KvAP (Jiang et al, 2003 Nature, 423). The SynK gene has been cloned in the shuttle vector pEGFP-1, suitable for expression of a GFP-tagged fusion protein in eukaryotic systems. The successful expression of a protein with the predicted molecular mass was revealed by Western blot and plasmamembrane targeting of the fusion protein in mammalian cell lines was verified by fluorescence microscopy. Patch clamp experiments pointed to functional expression. Concerning chloroplast channels, the molecular identity of most chloroplast envelope membrane channels is still unknown despite electrophysiological characterization of native membrane vesicles and application of proteomic approach. We identified by bioinformatic tools two putative ion channels which have a predicted transit peptide for chloroplasts and show good sequence homology with corresponding putative channels of Synechocystis. The two channels are members of the ClC chloride channel and of the glutamate receptor families, respectively. Biochemical analysis revealed the presence of both proteins in the envelope membranes of spinach chloroplasts. Furthermore, the effect of specific channel inhibitors on oxygen evolution in isolated chloroplasts indicated that these channels may contribute to the regulation of photosynthetic activity.
Email: ildi@civ.bio.unipd.it
Address: Dipartimento di Biologia, Università di Padova,, viale G. Colombo 3., 35121 Padova, Italy