Plasma membrane isolation from freshwater and salt-tolerant species ofChara: antibody cross-reactions and phosphohydrolase activities

Abstract

Plasma membranes were isolated using the aqueous polymer two-phase partition method from the algae Chara corallina and Chara longifolia, algae which differ in their ability to grow in saline environments. Enrichment of plasma membrane and depletion of tonoplast relative to the microsomal fraction was monitored using phosphohydrolase assays and cross-reactions to antibodies raised against higher plant transporters. Antibodies to the vacuolar ATPase and pyrophosphatase cross-reacted with epitopes in the microsomal fraction, but showed little affinity for the plasma membrane fraction. Pyrophosphatase activity also declined in the plasma membrane fraction relative to the microsomal fraction. The V-type H(+)-ATPase activity, sensitive to nitrate or bafilomycin, was low in both fractions, though the cross-reaction to the antibody was reduced in the plasma membrane fraction. By contrast, the antibody recognition of a P-type H(+)-ATPase amino acid sequence from Arabidopsis did not occur strongly in the anticipated 90-100 kDa range. While there was enhanced recognition of a polypeptide at around 140 kDa in the plasma membrane fraction, salt treatment of Chara longifolia resulted in plasma membrane fractions with reduced amounts of this epitope, but no change in vanadate-sensitive ATPase activity, suggesting that it does not represent the only P-type ATPase. Microsomal membranes from salt-adapted C. longifolia have higher reactivity with the antibody to the tonoplast ATPase.

Plasma Membrane Domains Participate in pH Banding of Chara Internodal Cells

“…These findings are consistent with earlier studies which showed the involvement of a vanadate-sensitive P-type ATPase in creating H + extrusion areas (Smith 1988, Mimura et al 1993, Badger and Price 1994, Ray et al 2003). So far, a characean P-type ATPase has been biochemically characterized only in one study in which an antibody from Arabidopsis recognized a 140 kDa polypeptide in Western blots of the salt-tolerant C. longifolia (Faraday et al 1996). Its molecular weight is higher than that measured for most proton pumps, suggesting the occurrence of several isoforms in plasma membranes of internodal cells.…”

Combining Experimental and Predicted Datasets for Determination of the Subcellular Location of Proteins in Arabidopsis

“…Often density and size either alone or together cannot reasonably be expected to cleanly separate membrane systems and organelle structures from each other. Techniques such as freeflow electrophoresis separation of membranes and organelles on the basis of charge (Bardy et al, 1998), phase partitioning of membranes (Rochester et al, 1987;Faraday et al, 1996), or isolation on the basis of immunoaffinity (Burgess and Thompson, 2002) are required. Combining density, size, charge, and affinity techniques will inevitably greatly reduce the yield of subcellular fractions, but may substantially increase purity for subcellular proteome analysis.…”

The role of ion‐transporting proteins in the evolution of salt tolerance in charophyte algae

“…This hypothesis was supported by the presence of a novel P‐type ATPase occurring only in the C. longifolia plasma membrane, and only under salt culture (Faraday et al. 1996). In addition to mechanisms that can directly assist in salt tolerance, aquaporins are a key driver in plant–water relations because they facilitate the movement of water across plant membranes, thus controlling the rate of change of turgor during turgor regulation.…”