Thylakoid membranes coupling factor

Moroney, J.V., Wamcke, K., and McCarthy, R.F. (1982) The distance between thiol groups in the gamma subunit of coupling factor 1 influences the protein permeability of thylakoid membranes. J. Bioenerg. Biomembr. 14, 347. 

Like mitochondria, chloroplasts (when illuminated) pump protons across their membranes (Fig. 23-18). However, while mitochondria pump protons to the outside, the protons accumulate on the inside of the thylakoids. The ATP synthase heads of coupling factor CEj are found on the outside of the thylakoids, facing the stromal matrix, while those of F, lie on the insides of mitochondrial membranes. However, the same mechanism of ATP formation is used in both chloroplasts and mitochondria (Chapter 18). 

Coupling between the H+ movements across the thylakoid membranes associated with electron flow and ATP formation occurs via a coupling factor known as an ATP synthetase, which is usually referred to as ATP synthase but also as an ATPase (because it can catalyze the reverse reaction leading to ATP hydrolysis). As illustrated in Figure 6-5, the ATP synthase has two components (1) a five-protein factor that occurs on the stromal side of a thylakoid, which can bind ADP, Pj, and ATP (labeled CFX in Fig. 6-5) and (2) a four-protein factor that is hydrophobic and hence occurs in the thylakoid membrane, through which H+ can pass (labeled CF0).5... 

CFi was originally isolated as a coupling factor, that is, a protein which when removed from the thylakoid membrane leaves a membrane unable to catalyse photophosphorylation, and which when reconstituted into it restores this ability. The... 

KR Miller and LA Staehelin (1976) Analysis of the thylakoid outer surface coupling factor is limited to unstacked membrane regions. J Cell Biol 68 30-47... 

The energy stored In this state Is consumed only along the selected modes, coupled with the ATP synthesis. This Is the essence of the concept of the "protein-machine" (10,12). The results of experiments on the ATP synthesis by Isolated coupling factors from chloroplasts, and of similar studies with some other H+-ATPases (13,14), allow a new approach to the experiments on phosphorylation Induced by pH-shift in the suspension of thylakoid membranes. This approach Is considered In accompaning paper (17). Some results described In this paper have been published In Russian earlier (15,16). 

Under non-energized conditions the coupling factors (CF-j) of dark-adapted thylakoid membranes are catalytically inactive and therefore incapable of even the thermodynamically favorable hydrolysis of ATP, Although the energetic requirement for... 

In order to test if the AF preparation was reconstitutively active, AFj- and CF -deficient membranes were prepaid by NaBr-treatment, and after the addition of AF or CFi to the deficient membranes, the recovery of photophosphorylation was measured (Table 2). The addition of AFj to the NaBr-treated thylakoids from A. nidulans resulted in recovery of more than 80% of the photophosphorylation of the non-treated particles. CFi from spinach restored photophosphorylation to the AFj-deficient membranes although to a lesser extent than AFj. Tlie reverse was true when the coupling factor activities were tested in CFj-deficient thylakoids from spinach. 

Energization of thylakoid membranes and coupling factor activity in intact leaf tissue can be assessed from analysis of the flash-induced... 

First, it was necessary to see if mature green-maize chloroplast membranes were coated with 90-A particles. Thylakoids were prepared and examined by negative staining in the electron microscope. On the surface of these membranes, we could see 90 A-particles, similar to those on spinach thylakoids and on maize etioplast membranes. This supported our opinion that the 90-A particles in etioplast membranes are identical to the coupling factor. However, some additional experiments were necessary to demonstrate this. 

M NaCl-0.15 mM EDTA and collecting the membranes by centrifugation. The stripped membranes could not carry on A/B phosphorylation. The capacity to do this was, however, restored by incubating coupling-factor preparations from chloropiasts or etioplasts of maize with stripped thylakoids in 0.01 m EDTA-0.1 m MgCl2. This showed that the CFi was required, but was probably not the limiting factor since it is present in etioplasts. What other conditions are required ... 

Fig. 6. Osmotic response and A/B phosphorylation of thylakoid and etioplast membranes in media of varying sucrose concentrations. Green A/B A/B phosphorylation of thylakoids. Etiolated A/B no detectable A/B phosphorylation in etioplast membranes. Green PPV osmotic response of thylakoid membranes. Etiolated PPV osmotic response of etioplast membranes. Green EDTA/PPV osmotic response of thylakoids extracted with EDTA-NaCl to remove coupling factor. The packed pellet volume in sucrose-free solution was taken as 100%. A/B activity was measured by assaying the amount of Pi incorporated into ATP. (From Forger and Bogorad. )...

The H" -ATPase of thylakoids catalyzes ATP synthesis and, in some conditions, hydrolysis in a manner that is linked to proton fluxes. The H+-ATPase consists of two readily separable parts coupling factor 1 (CF ), a hydrophilic, multisubunit enzyme that is extrinsic to the membrane, and Fq, a collection of more hydrophobic proteins that are intrinsic to the membrane. CF contains the active sites of the H -ATPase and is composed of five different subunits, labeled a-e in order of decreasing molecular weight. Fq constitutes a proton channel and provides specific sites for the attachment of CF. to the membrane. 

DCCD and triphenyltin chloride are less inhibitory of flash-induced ATP synthesis than of ATP synthesis driven by continuous illumination, which implies that the protonmotive force cannot be localized entirely between particular sites of proton generation and consumption. This idea is supported by the observation that flash-induced phosphorylation associated with different electron transport pathways was equally sensitive to subsaturaing amounts of triphenyltin, since there is less than one CF per complete electron transport chain, and coupling factors are apparently confined to non-appressed regions of the thylakoid membrane. 

Selman BR, Durbin RD (1978) Evidence for a catalytic function of the coupling factor 1 protein reconstituted with chloroplast thylakoid membranes. Biochim.Biophys. Acta 502, 29-37... 

As a coupling factor of photophosphorylation any protein can be defined which is isolated from chloroplasts and reconstitutes uncoupled thylakoid membranes. The treatment for resolution usually is incubation in low salt in the presence of EDTA. A coupling factor purified from such extract need not have catalytic activity, even at the membrane, but may be required for regulation of H flux or for binding of the peptides containing the active center of the ATPsynthase. Such a role was ascribed to the 5 peptide of CF, since re-addition of S is necessary for reconstitution (Nelson,Karny,1976 Younis et al. 1977 Roos, Berzborn, 1982, Andreo et al. 1982). 

The salt washed thylakoid membranes from spinach chloroplasts were treated with a primary amine-specific fluorescent chemical modifier, Fluorescamine (4-Phenylspiro furan-2 lQ 1-phthalan 3t3 dione). The effects of this covalent chemical modification on various processes of energy transduction were studied. It is suggested that the chemical modification by fluorescamine of free amino group of coupling factor 1 results in different conformations of CFi ... 

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