F type ATPase

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. 

F-Type ATPases Are Reversible, ATP-Driven Proton Pumps... 

FIGURE 11-39 Structure of the F F] ATPase/ATP synthase. F-type ATPases have a peripheral domain, F, consisting of three cr subunits, three j3 subunits, one S subunit (purple), and a central shaft (the y subunit, green). The integral portion of F-type ATPases, F0 (yellow), has multiple copies of c, one a, and two b subunits. F0 provides a transmembrane channel through which about four protons are pumped (red arrows) for each ATP hydrolyzed on the j3 subunits of F,. The remarkable mechanism by which these two events are coupled is described in detail in Chapter 19. It involves rotation of F0 relative to F, (black arrow). The structures of V0Vi and AoA, are essentially similar to that of F0F, and the mechanisms are probably similar, too. 

FIGURE 11-40 Reversibility of F-type ATPases. An ATP-driven proton transporter also can catalyze ATP synthesis (red arrows) as protons flow down their electrochemical gradient. This is the central reaction in the processes of oxidative phosphorylation and photophosphorylation, both described in detail in Chapter 19. 

The reaction catalyzed by F-type ATPases is reversible, so a proton gradient can supply the energy to drive the reverse reaction, ATP synthesis (Fig. 11-40). When functioning in this direction, the F-type ATPases are more appropriately named ATP synthases. ATP synthases are central to ATP production in mitochondria during oxidative phosphorylation and in chloroplasts during photophosphorylation, as well as in eubacteria and archaebacteria. The proton gradient needed to drive ATP synthesis is produced by other types of proton pumps powered by substrate oxidation or sunlight. As noted above, we return to a detailed description of these processes in Chapter 19. 

P-type ATPases 398 SERCA pump 400 F-type ATPases 401 ATP synthase 401 V-type ATPases 401 ABC transporters 402 multidrug transporter 402... 

Capaldi, R. A., Aggeler, R., Turina, P., and Wilkens, S. (1994). Coupling between the catalytic sites and the proton channel in 1) F,-type ATPases. TIBS 219, 284—289. 

An H+ electrochemical gradient (ApH+) provides the energy required for active transport of all classical neurotransmitters into synaptic vesicles. The Mg2+-dependent vacuolar-type H+-ATPase (V-ATPase) that produces this gradient resides on internal membranes of the secretory pathway, in particular endosomes and lysosomes (vacuole in yeast) as well as secretory vesicles (Figure 3). In terms of both structure and function, this pump resembles the F-type ATPases of bacteria, mitochondria and chloroplasts, and differs from the P-type ATPases expressed at the plasma membrane of mammalian cells (e.g., the Na+/K+-, gastric H+/K+-and muscle Ca2+-ATPases) (Forgac, 1989 Nelson, 1992). The vacuolar and F0F1... 

The V-type (or vacuolar-type) H+-ATPase was first found in fungal vacuoles and plant tonoplasts,13 l4) and is now known to be widely distributed in mammalian endomembrane systems.15) Like F-type ATPase, the subunit structure of the V-type ATPase is complicated. The membrane extrinsic sector is composed of 73 kDa, 58 kDa, 38 kDa, and 34 kDa subunits, while the membrane intrinsic sector is composed of 40 kDa 20 kDa and 16 kDa subunits. 

In this chapter, we discuss our current understanding of the structure and mechanism of the F-type ATPase. However, a comprehensive review is not intended, and the results discussed are mainly from our recent studies on E. coli enzyme. Naturally discussion for important results from mitochondria or chloroplasts are included. For other aspects of the enzyme, readers are referred to excellent review articles that have appeared recently.3 10 ... 

Structure and Function of F-type ATPase 11.2.1 Preparation of FoFi... 

V-type and P-type ATPases are present in the archaea. Although the V-like archaeal ATPases have been asserted to be ATP synthases [4,43] the evidence for this is at best inferential [44]. The ATPase from H. saccharovorum is proposed to be an F-type ATPase on the basis of the mechanism of ATP hydrolysis [44]. However, there is increasing evidence that the archaea possess a variety of ATPases, and with the possible exception... 

The best evidence for the existence of an F-type ATPase in the archaea comes from experiments with a methylotropic methanogen [52], Membranes contain particles that react with polyclonal antiserum against the 3 subunit of the Escherichia coli F-ATPase. In addition, negative staining reveals particles attached to the membranes with a stalk whose appearance is suggestive of an F-type ATPase [53]. Unfortunately, no further studies have... 

The membrane-bound ATPase from M barkeri is inhibited by DCCD and the inhibitor is bound to a small hydrophobic peptide (Mr 6000). A DCCD-sensitive ATPase, solubilized by octylglucoside, has been purified in the presence of the detergent. This form of the ATPase contains six subunits (Mr 62000, 49000, 40000, 27000, 23000, and 6000). When incubated with C-DCCD, all the radioactivity is associated with the Mr 6000 subunit. [56]. This subunit is smaller than the DCCD-binding peptides from S. acidocaldarius [57] and coated vesicles [58]. The function of the Mr 6000 peptide has yet to be establishe4 although its properties suggest that it may be equivalent to the subunit c, the DCCD-binding peptide of F-type ATPases. 

The membrane-bound and purified enzymes lose little activity over a 2-3 month period when stored at room temperature in the presence of 4M NaCl. The purified ATPase is rapidly inactivated at 4°C and this is due to the dissociation of the enzyme [23]. The H. saccharovorum ATPase is more active in NaCl than in KCl, and most active when assayed between pH9 and 10 in the presence of at least 3.5M NaCl [19]. The membrane-bound enzyme rapidly loses activity when stored in 400 mM NaCl but is considerably more stable when either spermine (30 mM) or MgCl2 (100 mM) is present [34]. The purified ATPase is rapidly inactivated when stored in IM NaCl but is as stable in 500mM NaCl/lM (NH4)2S04 as in 4 M NaCl. The halobacterial ATPase is not inhibited by vanadate, azide, or Dio-9 [19] but is inhibited by DCCD when the enzyme is preincubated at pH 6 or less and at relatively high quantities of DCCD (125 nmol/mg protein). When membranes are incubated with C-DCCD, virtually all the radioactivity is associated with subunit II [19]. The conditions of inhibition are similar to those that inhibit F] activity [72-74]. This suggests that subunit II may be similar to the (3 subunit of F-type ATPases, an assumption consistent with the immunological reactivity of the subunit. Antiserum against the 3-subunit from the ATPase from 5. acidocaldarius which reacts with the 3 subunit from F-type ATPases [51] also reacts with subunit II from H. saccharovorum [60]. 

The properties of the H. salinarium halobium) enzyme are those expected of a V-type ATPase. Polyclonal antibodies prepared against the H. salinarium halobium) ATPase react with the two largest subunits from the ATPases from S. acidocaldarius and beet root tonoplast, but fail to react with the subunits from chloroplast F-ATPase [87]. This result contradicts the observation that polyclonal antiserum against the 3 subunit from S. acidocaldarius, which reacts with various 3 subunits from F-type ATPases, reacts with subunit II from H. saccharovorum and a M, 67000 subunit from the H. salinarium halobium) ATPase [60]. 

Like mitochondria, chloroplasts have an F-type ATPase which generates ATP from a proton gradient. Thylakoids are permeable to Mg2+ and Cl" so this is mostly a concentration effect (pH) rather than an electrical (charge separation) effect. In fact, the pH difference can be very marked typically the lumen is at pH 4 and the stroma at pH 8. 

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