In many conditions, cells require solutes in quantities that cannot be achieved through passive transport. Similarly, many waste products need to be removed or compartmentalised often against their electrochemical potential. This transport can, by definition, not occur via passive transport and requires active systems that directly use energy. A further fundamental requirement of all living cells is the capacity to convert chemical energy into electrochemical energy and vice versa. Primary active transport mechanisms convert metabolic energy to move substrates against a gradient. At the same time, many primary systems, through their activity, energise membranes by establishing electrochemical potential differences. The latter can subsequently be used to energise secondary active transport (Section 5.4).
The majority of chemical energy that is generated from processes such as respiration and photosynthesis is deposited in the phospho-ester bonds of compounds like adenosine triphosphate (ATP), guanyl triphosphate (GTP) and pyrophosphate (PPi). Hydrolysis of the phosphate-ester bond is highly exergonic; for example the conversion of ATP to adenosine diphosphate (ADP) has a AG° of -32 kJmol-1. This energy can be converted into other forms such as an electrochemical gradient. In plant cells, a large amount of ATP is used for transmembrane H+ movement. Extrusion of H+ from the cytoplasm has physiological rationales; for example, for cellular pH regulation, the acidification of cell walls and rhizosphere, or to lower the pH in lytic compartments. Nevertheless, the principal function of plant H+ ATPases, or H+ pumps, is in the generation of transmembrane H+ gradients. The pH and electrical potential differences of the H+ gradient can both be used in subsequent transport processes.
Plant proton ATPases, as most other pumps, have very low turnover rates of ~100 H+ per second. This and their crucial role in cellular physiology mean proton ATPases are very prolific enzymes that can make up several percent of total protein. They are predominantly found in the plasma membrane and tonoplast and also in other endomembrane systems such as ER and Golgi complex (see also Chapter 7). Often the level of expression of these pumps is related to physiological conditions and function of tissues and cell types.
The proton pump located in the plasma membrane is a P-type ATPase (see Geisler et al. (1999), Sze et al. (1999) and Axelsen and Palmgren (2001) for reviews) so called because during its catalytic cycle, phosphorylation of a specific site at the enzyme is essential (Colour plate 5.6). In plants this enzyme generates a proton motive force (PMF) across the plasma membrane, typically in the region of 250-300 mV comprising a membrane potential of around -150 mV (negative inside the cell) and a ApH of around 2 units (acidic outside the cell), which is equivalent to -120 mV. Both components of the PMF are used to energise movement of many important nutrients and metabolites through active H+ -coupled transport. In addition, the activity of P-type ATPases is a dominant contributor to membrane polarisation and therefore impacts greatly on the driving force for passive transport.
The AG° for ATP hydrolysis is around 32 kJ mol-1. This would be equivalent to around -330 mV if it is assumed that 1 ATP is hydrolysed per H+ pumped, since AG° = -nFAE°■ with the stoichiometric coupling ratio n equal to unity, a conversion factor between mV and kJ mol-1, the Faraday constant F, of 96.5 kJ V-1 mol-1 and E° the electropotential in V. However, in physiological conditions, the AG for ATP hydrolysis is considerably higher and in the region of 50 kJmol-1, theoretically sufficient to generate a PMF of over -500 mV if n were 1. Experiments where H+ fluxes were compared with ATP hydrolysis rates indeed showed that 1 H+ is pumped for every ATP hydrolysed.
P-type proton pumps are encoded by multiple-gene families and the enzyme functions in the membrane as a single polypeptide of around 100 kDa. Structurally, P-type pumps contain 10 TMD with the TMD4-TMD5 cytoplasmic loop containing the ATP binding site. The same loop also has a conserved aspartyl residue that becomes phosphorylated and dephosphorylated during every catalytic cycle. Toxins such as vanadate and arsenate mimic phosphate and inhibit the enzyme by binding to the phosphorylation site. The C-terminal residues of the protein function as an autoinhibitory domain. Cleavage of this ~100 amino acid tail by trypsin, or expression of the shortened mutant protein, leads to activation of the enzyme. The same domain also interacts with 14-3-3 proteins that can bind to it and, through unknown mechanisms, activates the ATPase (Colour plate 5.6). The fungal toxin fusicoccin is believed to stabilise the interaction between ATPase and 14-3-3 and thus activate the protein.
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