K+-conducting ion channel of the chloroplast inner envelope: functional reconstitution into liposomes.

Abstract

Potassium flux between the chloroplast stroma and cytoplasm is known to be indirectly linked to HI countertransport and, hence, stromal pH and photosynthetic capacity. The specific molecular mechanism that facilitates K+ flux across the chloroplast envelope is not known and has been a source of controversy for well over a decade. The objective of this study was to elucidate the nature of this envelope protein.To this end, solubilized protein in detergent extracts of purified chloroplast inner envelope vesicles was reconstituted into artificial liposomes, and cation fluxes into these proteoliposomes were measured. Results of inhibitor studies and counterflux experiments indicated that a K+-conducting ion channel was solubilized and functionally reconstituted into the proteoliposomes. This transport protein may be a nonspecific monovalent cation channel. This report represents a direct demonstration of ion channel activity associated with the limiting (inner) membrane of the chloroplast envelope.Photosynthesis is critically dependent on the maintenance of high stromal pH in the illuminated chloroplast (1) due to the high pH optima of several photosynthetic carbon reduction cycle enzymes. This important regulatory phenomenon necessitates development of a pH gradient between the illuminated stroma (at pH 8) and both the thylakoid lumen (pH < 6) and the external medium (i.e., cytosol, at pH 7) in situ.For well over a decade it has been known that H+ flux across the chloroplast envelope is indirectly linked to K+ countercurrents, which likely occur through a separate (wellregulated) uniport pathway (2). Inhibitor studies with intact isolated chloroplasts further suggest that trans-envelope K+ flux may occur through a putative ion channel (2-4).Characterization of this K+ transport pathway, however, has not been possible because of the nature of the experimental system (i.e., the intact chloroplast) used in previous work. Definitive conclusions about this putative ion channel await the development of an experimental system-i.e., extraction of the protein from its native membrane, maintenance of itsfunctional integrity, reconstitution into a homogenous membrane system (e.g., artificial liposomes), and development of a transport assay sensitive enough to measure the specific activity of the protein. These objectives were the primary focus of the experiments described in this report. The significance of the work is underscored by the following: (i) the existence of ion channels in the inner membrane of the chloroplast envelope is demonstrated-the biophysical nature of the envelope cation transporter has been the subject of speculation for well over a decade (5, 6); and (ii) relatively little is known about K+ channels in general-molecular characterization of this class of proteins is just beginning and purification of a functional K+ channel from any (plant or animal) source has not yet been reported (7).The publication costs of this article were defrayed in part by page charge payment. This article must therefore...

Characterization of a Chloroplast Inner Envelope K+ Channel

“…After microcentrifugation (in a Beckman Microfuge B, used because the tubes are held perpendicular to the rotor ais), radioisotope retained on the filter was counted (Beckman 3801 liquid scintillation counter) using dual-label dpm programs with quench correction. The [3H]-mannitol retained on the filter, which is not taken up into the osmotic volume of the vesicles (Wang et al, 1993), allowed for background correction within each sample of Rb' retained on the filter but not taken up by vesicles. For a11 experiments, four tubes served as replicates for each treatment.…”

“…The activity of functional K+-conducting channel proteins in native chloroplast jnner-envelope membrane vesicles and after reconstitution of detergent-extracted protein into artificial liposomes was evaluated using a sensitive microcentrifugation/filtration assay developed previously in this laboratory (Berkowitz and Peters, 1993). As described previously, this flux assay involves construction of a filtration device with a 0.22-pm pore filter sandwiched between the cut end of a I-mL plastic syringe (serving as an incubation reservoir) and a 400-pL microcentrifuge tube.…”

“…Inner-envelope vesicles were prepared from spinach (Spinacia oleracea L.) as described previously (Berkowitz and Peters, 1993). Briefly, intact chloroplasts (isolated from 5-10 kg of spinach leaves using Perco11 step gradients) were exposed to freeze/thaw cycles in hyperosmotic medium, and the thylakoids were removed before the crude membrane fraction was loaded on a discontinuous SUC step gradient.…”

“…Procedures for reconstitution of detergent-extracted envelope protein into enlarged liposomes followed those described by Wang et al (1993). Detergent-extracted protein was mixed with 20 mg/mL homogenized and sonicated asolectin stock at a ratio of 2.4:1.08 (v/v).…”

“…Movement of K+ through this channel protein, therefore, has a profound effect on stromal pH and, hence, photosynthesis (Werdan et al, 1975;Maury et al, 1981; Wu and Berkowitz, 1992). Previous work from this laboratory has demonstrated that a K'-conducting protein can be successfully detergent-solubilized from preparations of purified inner-envelope membrane vesicles and functionally reconstituted into artificial liposomes (Wang et al, 1993). This putative K+ channel protein has not previously been characterized.…”