Evidence for the functioning of a Cl−/H+ antiporter in the membranes isolated from root cells of the halophyte Suaeda altissima and enriched with Golgi membranes

Abstract

Cl−/H+ exchange activity in the membranes isolated from the root cells of the halophyte Suaeda altissima (L.) Pall. was originally revealed and characterized. The membrane vesicles were isolated by centrifugation of microsomes in a continuous iodixanol density gradient. The highest activity of latent inosine phosphatase, a marker of Golgi membranes, was localized in the upper part of the gradient, indicating its enrichment with Golgi membranes. The same part of the gradient was characterized by the highest Cl−/H+ exchange rate. The Cl−/H+ exchange activity was detected as electrogenic ΔpCl-dependent H+ transport monitored as changes in differential absorbance of a ΔpH-probe acridine orange, or as changes in fluorescence excitation spectrum of a pH-probe pyranine loaded into the vesicles. Generation of transmembrane electric potential (Δψ) during the Cl−/H+ exchange was assayed as changes in differential absorbance of a Δψ-probe safranin O. Establishing the transmembrane ΔpCl inward vesicles resulted in H+ efflux sensitive to DIDS (4,4′-diisothiocyano-2,2′-stylbene-disulfonic acid), an inhibitor of chloride transporters and channels, and generation of Δψ negative inside. To maintain the ΔpCl-dependent H+ efflux from the vesicles, either the presence of a penetrating cation tetraphenylphosphonium neutralizing negative charges inside the vesicles or null K+ diffusion potential across the membranes was required. The results demonstrate the activity of an electrogenic Cl−/H+ antiporter in the fraction enriched with Golgi membranes. We hypothesize that the Cl−/H+ antiporter is involved into the regulation of cytoplasmic Cl− concentrations by vesicular trafficking of Cl− from the cytoplasm to the vacuole by endosomes, derivatives of Golgi membranes.