ATPases uptake function

ATP-dependent uptake of copper into everted membrane vesicles from cells expressing ecCopA could be demonstrated. Transport was inhibited by the classical P-type ATPase inhibitor vanadate. Dithiothreitol, a strong reductant, was required for ecCopA-catalyzed Cu uptake, suggesting that the substrate of ecCopA is Cu(l). Thus the function of ecCopA resembles that of the En. hirae CopB ATPase by functioning as a copper efflux pump in vivo when excess copper is present in the cytoplasm (Rensing et al., 2000). 

Figure 12.2 Copper chaperone function, (a) Copper homeostasis in Enterococcus hirae is affected by the proteins encoded by the cop operon. CopA, Cu1+-import ATPase CopB, Cu1+-export ATPase CopY, Cu1+-responsive repressor copZ, chaperone for Cu1+ delivery to CopY. (b) The CTR family of proteins transports copper into yeast cells. Atxlp delivers copper to the CPx-type ATPases located in the post Golgi apparatus for the maturation of Fet3p. (c) Coxl7p delivers copper to the mitochondrial intermembrane space for incorporation into cytochrome c oxidase (CCO). (d) hCTR, a human homologue of CTR, mediates copper-ion uptake into human cells. CCS delivers copper to cytoplasmic Cu/Zn superoxide dismutase (SOD1). Abbreviations IMM, inner mitochondrial membrane OMM, outer mitochondrial membrane PM, plasma membrane PGV, post Golgi vessel. Reprinted from Harrison et al., 2000. Copyright (2000), with permission from Elsevier Science.

Calmodulin, a calcium binding protein, is involved in Ca2+-dependent regulation of several synaptic functions of the brain synthesis, uptake and release of neurotransmitters, protein phosphorylation and Ca+2 transport. It reacts with TET, TMT and TBT which then inactivates enzymes like Ca+2-ATPase and phosphodiesterase. In vitro studies indicated TBT was greater at inhibiting calmodulin activity than TET and TMT, whereas in vivo the order was TET > TMT > TBT. This may be due to the greater detoxification of TBT (66%) in the liver before moving to other organs30,31. 

F-ATPases (including the H+- or Na+-translocating subfamilies F-type, V-type and A-type ATPase) are found in eukaryotic mitochondria and chloroplasts, in bacteria and in Archaea. As multi-subunit complexes with three to 13 dissimilar subunits, they are embedded in the membrane and involved in primary energy conversion. Although extensively studied at the molecular level, the F-ATPases will not be discussed here in detail, since their main function is not the uptake of nutrients but the synthesis of ATP ( ATP synthase ) [127-130]. For example, synthesis of ATP is mediated by bacterial F-type ATPases when protons flow through the complex down the proton electrochemical gradient. Operating in the opposite direction, the ATPases pump 3 4 H+ and/or 3Na+ out of the cell per ATP hydrolysed. 

Ion transport in very dilute freshwater environments can also be problematic from the perspective of electrochemical theory. For example, electrochemical theory predicts that Na+ entry through epithelial channels will stop when the external Na+ is <0.1 mmol Na+ (typical of many freshwaters), given intracellular Na+ concentrations of 10-20 mmol 1 1 in gill epithelial cells [83], Thus elevation of Na+ concentrations at the epithelial surface (adsorption) becomes critical to ion-channel function and Na+ uptake into the cells [50], Alternatively, we might postulate other transporters coupled to the Na+ channel to drive uptake (e.g. H+ slippage [84] on the H+-ATPase). 

As we saw in Chapter 7, zinc uptake in plants involves proteins of the ZIP family, some of which are root specific while others are found in both roots and shoots. The transport of zinc from the cytosol in many organisms is often associated with members of the cation diffusion facility (CDF) family. Although there are 12 predicted family members in Arabidopsis, only one, MTP1, has been characterized, which seems to function in the transport of Zn into the vacuole. Two members of the heavy metal ATPase (HMA) family, HMA2 and HMA4, have been shown to function in the transport of zinc out of the cells across the plasma membrane. 

The sER also functions as an intracellular calcium store, which normally keeps the Ca level in the cytoplasm low. This function is particularly marked in the sarcoplasmic reticulum, a specialized form of the sER in muscle cells (see p. 334). For release and uptake of Ca " ", the membranes of the sER contain signal-controlled Ca channels and energy-dependent Ca ATPases (see p. 220). In the lumen of the sER, the high Ca " " concentration is buffered by Ca -binding proteins. 

Cyclopiazonic acid is a potent inhibitor of calcium uptake and acts as a selective inhibitor of the sarco-endoplasmic reticulum Ca2+ATPases (SERCAs) [53], it induces charge alteration in plasma membranes and mitochondria and it can function as an antioxidant [54]. 

The outer membrane, the plasmalemma, efficiently protects the cell from the environment while, at the same time, carrying out functions important for cell metabolism the uptake of substrates and the elimination of toxic compounds. Substrate exchange with the environment is controlled by transport proteins embedded in the membrane (energy-requiring pumps such as Na+,K+-ATPase, or other transport units such as the Na+/glucose cotransporter and sodium and calcium ion channels) [1],... 

Figure 10. Experiment demonstrating consecutive dissociation of two calcium ions from the uptake sites of the SR Ca2+-ATPase in the unphosphorylated E, state. 45Ca2+ bound from the cytoplasmic side at the two high-affinity sites on the enzyme can be seen to dissociate back to the medium on the cytoplasmic side in two distinct phases upon dilution of the radioactivity (A). The first phase is rapid and independent of the concentration of Ca2+ in the medium. The second phase is also rapid in the absence of Ca2+ (presence of EGTA) in the buffer medium (open triangles) but slow in the presence of nonradioactive Ca2+ in the buffer medium (closed triangles, 10 pM Ca2+ open circles, 30 pM Ca2+ closed circles, 100 pM Ca2+ open squares, 300 pM Ca2+ closed squares 1 mM Ca2+). The inset shows the rate constant for dissociation of the second Ca2+ as function of the Ca2+ concentration in the medium. The lower panel (B) explains these observations in terms of steric hindrance of the dissociation of the deeper ion by the presence of a more superficial ion at its binding site (closed circles, radioactive 45Ca2+ open circles, nonradioactive 40Ca2+ from the medium). Reproduced from Orlowski and Champeil, 1991a with permission from The American Chemical Society.

Copper ion homeostasis in prokaryotes involves Cu ion efflux and sequestration. The proteins involved in these processes are regulated in their biosynthesis by the cellular Cu ion status. The best studied bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae. Cellular Cu levels in this bacterium control the expression of two P-type ATPases critical for Cu homeostasis (Odermatt and Solioz, 1995). The CopA ATPase functions in Cu ion uptake, whereas the CopB ATPase is a Cu(I) efflux pump (Solioz and Odermatt, 1995). The biosynthesis of both ATPases is regulated by a Cu-responsive transcription factor, CopY (Harrison et al., 2000). In low ambient Cu levels Cop Y represses transcription of the two ATPase genes. On exposure to Cu(I), CopY dissociates from promoter/operator sites on DNA with a for Cu of 20 jlM (Strausak and Solioz, 1997). Transcription of copA and copB proceeds after dissociation of CuCopY. The only other metal ions that induce CopY dissociation from DNA in vitro are Ag(I) and Cd(II), although the in vivo activation of copA and copB is specihc to Cu salts. The CuCopY complex is dimeric with two Cu(I) ions binding per monomer (C. T. Dameron, personal communication). The structural basis for the Cu-induced dissociation of CopY is unknown. Curiously, CopY is also activated in Cu-dehcient cells, but the mechanism is distinct from the described Cu-induced dissociation from DNA (Wunderh-Ye and Solioz, 1999). 

---