The plasma membrane H-ATPase forms a phosphorylated intermediate during ATP hydrolysis and
belongs to the P-ATPase family [92]. Similar intermediates are formed by other ion-transporting P-AT-
Pases. These ATPases are specifically inhibited by orthovanadate ions. The plasma membrane H-AT-
Pase has a functional molecular mass of 200 kDa and is apparently composed of two 100-kDa subunits,
each forming at least 8, and possibly 10, transmembrane helices [93]. Optimal pH for this enzyme is 6.5;
its activity is enhanced (maximally doubled) by K, but the enzyme is not directly involved in Ktrans-
port [94]. The H-ATPase is a major component of the plasma membrane. In root cells with high trans-
port activity, there are about 10^6 molecules of the enzyme per cell, with a turnover number of 20–100
sec^1. This results in proton fluxes of 10–100 pmol sec^1 cm^2 [95].
The tonoplast H-ATPase of plant cells does not form a phosphorylated intermediate and is not in-
hibited by vanadate. It belongs to the family of V-ATPases, operating as proton pumps at endomembranes
of eukaryotic cells [91]. The vacuolar H-ATPase is inhibited by the antibiotic bafilomycin [96] and by
NO 3 . It is stimulated by Cland not by monovalent cations; the pH optimum is 7.9 [92]. A functional
molecular mass of 750 kDa was assigned to the enzyme, which is composed of at least 10 different sub-
units [97] and exhibits four potential transmembrane helices [92].
Ca^2 -ATPase Another primary active carrier is the enzyme Ca^2 -ATPase. This enzyme is located in
the plasma membrane [98], in the tonoplast [99], and in the endoplasmic reticulum [100]. The Ca^2 -
ATPase forms a phosphorylated intermediate, is inhibited by vanadate [101,102], and belongs to the
P-ATPase family [103]. Ca^2 -ATPases transport Ca^2 out of the cytoplasm to the free space, into the vac-
uole, and into endoplasmic reticulum vesicles. Ca^2 -ATPases, together with Ca^2 /Hantiporters
(see Mineral Cations in Sec. V.B.3), are involved in Ca^2 homeostasis of the cytosol and regulate the
Ca^2 activity there at about 0.1 M [104]. Cytosolic Ca^2 [103] activity rises only transiently in response
to certain stimuli [105]. Alignment of eukaryotic Ca^2 -ATPases shows that the plant and animal genes
are related [99] and can be divided into two types of pumps, those that are stimulated by calmodulin and
those that are not [106]. Calmodulin is a calcium-modulated protein involved in many Ca^2 -regulated
processes [107].
A spinach plasma membrane Ca^2 -ATPase was identified as a 120-kDa polypeptide. Calmodulin in-
creased the Vmaxfor Ca^2 pumping into inside-out spinach plasma membrane vesicles (homologous to
pumping from the cytosol to the free space) threefold and decreased the Kmfrom 1.6 to 0.9 mM. During
trypsin treatment (limited proteolysis) the amount of the 120-kDa polypeptide diminished and a 109-kDa
polypeptide appeared. The appearance of the 109-kDa polypeptide correlated with increased enzyme ac-
tivity and loss of calmodulin regulation. Limited proteolysis increased the Vmaxfor Ca^2 pumping more
than calmodulin [108]. The contribution of the Ca^2 -ATPase to the electrical potential difference across
the plasma membrane is insignificant because its activity is two orders of magnitude lower than that of
the H-ATPase [95].
Three genes encoding calmodulin-stimulated Ca^2 -ATPases have been cloned from plants, ACA1
andACA2fromArabidopsis thalianaandBCA1fromBrassica oleracea. None localized to the plasma
membrane; they are expressed in endomembranes and the tonoplast [109]. A further three genes en-
coding calmodulin-insensitive Ca^2 -ATPases have been cloned from plants, LCA1fromLycopersicum
esculentum, pH27fromNicotiana tabacum, and ACA3(ECA1) from Arabidopsis thaliana. LCA1was
localized to the tonoplast and the plasma membrane and both pH27andACA3to endomembranes
[109].
ABC TRANSPORTERS An additional group of primary active solute transporters have been demon-
strated. They belong to the ABC (ATP-binding cassette) superfamily [110]. These transporters were orig-
inally identified in bacterial and animal cells. The ABC transporters use Mg^2 -ATP as a direct energy
source for transport; they form a phosphorylated intermediate during hydrolysis and belong to the P-type
ATPases. All ABC transporters consist of an integral membrane sector and a cytosol-orientated ATP
binding domain. The membrane sector of the ABC transporters provides the pathway for transport and
determines the molecular specificity of the transporter.
Two major subclasses of ABCs have been molecularly identified in plants: MRPs (multiple drug re-
sistance–associated proteins) and MDRs (multidrug resistance proteins, so named according to their ani-
mal prototypes). Only MRPs have been defined functionally. MRPs are localized to the vacuole and func-
tion in detoxification. A mineral ion–related function of MRPs is detoxification of Cd^2 and perhaps
other heavy metals. MRP functions in detoxification by transport of metal phytochelatins to the vacuole
346 JACOBY AND MORAN