Calcium and Proton Transport in Membrane Vesicles from Barley Roots

Abstract

(3,11,19,22,31,32) while the Ca2+-ATPases are associated with the ER and plasma membrane (6,7,9,11,17,28,31). It has been suggested that both types oftransporter may exist in a single membrane type (2, 30) although this has not been investigated in higher plants. We examined the Ca2+-transport activity of microsomes isolated from barley roots in order to determine which ofthe two types ofCa2+ transporters were associated with the major organelle fractions.The mechanisms by which Ca2+ is transported out of the cytoplasm against a large concentration gradient can be studied using isolated membrane vesicles (3,6,7,11,17,22,28,31,32 (13)(14)(15)(16). In the present paper the mechanisms and capacity for formation of Ca2`gradients by these vesicles are assessed. A prominent Ca2+/nH+ antiporter activity in tonoplast vesicles is characterized and evidence for a non-ER, non-plasma membrane Ca2+-ATPase is presented. A distinction is made between the activity of the Ca24/nH' antiporter and independent effects of Ca2+ on the formation and maintenance of ApH by the PM-and V-ATPases.Precise regulation ofcytoplasmic Ca2" may be an important element of signal transduction in plant cells (23,27), as it is in animal cells (12). Resting levels of cytoplasmic Ca2" are reported to be less than 0.5 AM in plants (8,18,26). These low levels ofcytoplasmic Ca2" are maintained by active uptake into organelles and export across the plasma membrane (7,12,26). To date, two types of Ca2" transport activity have been described in plant cells, one which is dependent on the MATERIALS AND METHODS Plant MaterialSeeds of barley (Hordeum vulgare L. cv CM72) were sown above an aerated solution containing full strength nutrients 2 Abbreviations: ApH, difference in pH across the vesicle membrane; ESR, electron spin resonance; PM-ATPase, plasma membrane H+-ATPase; PM, plasma membrane-enriched membrane fraction; 6,"5N,2,6,6, 1-'5N-1-oxyl; TN, tonoplast-enriched membrane fraction; Q, fluorescence quench; V-ATPase, vacuolar-type H+-ATPase; DCCD, dicyclohexylcarbodiimide; nH, number of H+ where n is unknown; BTP, bistris propane.

A Single Gene May Encode Differentially Localized Ca 2+ -ATPases in Tomato

“…The inhibitor sensitivity of Ca2+ uptake by TN vesicles suggested the presence of two Ca2+ transport systems operating in this membrane: one is the LCA Ca2+-ATPase, and the other is a Ca2+/H+ antiporter. The presence of both transporters in the same membrane has been reported previously in maize leaf PM (Kasai and Muto, 1990) and in the TN of barley roots (Dupont et al, 1990) and spinach leaves (Malatialy et al, 1988).…”

“…It has been proposed that Ca2+-ATPases are primarily responsible for setting resting levels of cytosolic calcium (Evans et al, 1991), whereas the lower affinity Ca2+/H+ antiporter may be more important during periods or locations of increased cytosolic Ca2+. Although a TN Ca2+-ATPase has been reported previously (Dupont et al, 1990;Gavin et al, 1993), most of the studies and discussions about the role of higher plant Ca2+-ATPases in cellular Ca2+ homeostasis have focused on only the ER and PM Ca2+-ATPases. However, the presence in the TN of a primary Ca2+-ATPase may be of particular significance in the regulation of cytoplasmic Ca2+ levels.…”

“…Moreover, there are conflicting reports concerning the calmodulin sensitivity of Ca 2+ -ATPases in plant ER and PM preparations (Evans, 1994), indicating the possibility of biochemical differences between species and/or organs. Reports characterizing plant vacuolar Ca 2+ -ATPases are less abundant, but their presence has been demonstrated in several plants (Malatialy et al, 1988;Zocchi, 1988;Dupont et al, 1990;Gavin et al, 1993).…”

Distinction between Endoplasmic Reticulum-Type and Plasma Membrane-Type Ca2+ Pumps (Partial Purification of a 120-Kilodalton Ca2+-ATPase from Endomembranes)

“…For example, a prominent CaMstimulated Ca2+ pump on the PM of radish seedlings hydrolyses ATP, GTP, or ITP (Rasi-Caldogno et al, 1992). However, it is not clear whether this pump is related to another CaM-stimulated Ca2+ pump that is localized to the tonoplast in barley or corn roots (DuPont et al, 1990;Gavin et al, 1993). The animal ER-type Ca2' pump is distinguished from the animal PM-type Ca2+ pump by its sensitivity to thapsigargin or cyclopiazonic acid, and by its insensitivity to CaM (Schatzmann, 1989;Siedler et al, 1989;Carafoli 1992); however, the results from plants are more ambiguous.…”

In Vitro and in Vivo Phosphorylation of Polypeptides in Plasma Membrane and Tonoplast-Enriched Fractions from Barley Roots

“…Because the assays for ion transport activity involve the addition of ATP to membrane fractions, it seemed likely that transport activities might be modulated by in vitro phosphorylation of the membrane proteins. For example, proton transport across the tonoplast and plasma membrane is differentially altered by including calcium in the proton transport assay (9); one explanation might be that addition of calcium stimulates a protein kinase in either membrane. Also, there are potential roles for in vivo 'Funded by Binational Agricultural Research Development Fund grant I-1075-86.…”