The Phosphate Carrier from Yeast Mitochondria

Schroers, Andreas; Burkovski, Andreas; Wohlrab, Hartmut; Krämer, Reinhard

doi:10.1074/jbc.273.23.14269

Cited by 80 publications

(29 citation statements)

References 49 publications

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“…In contrast, however, past biochemical studies suggest that the active form of ANC is a dimer (32,33). A dimeric form of PIC is also believed to be the active form from similar biochemical studies (34). However, each study has focused only on one transporter and not the two together, leaving open the possibility that a PIC/ANC heterodimer is really the physiological form.…”

Section: Discussionmentioning

confidence: 76%

Mitochondrial ATP Synthasome

Chen

Ko²,

Delannoy

et al. 2004

Journal of Biological Chemistry

190

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The terminal steps involved in making ATP in mitochondria require an ATP synthase (F 0 F 1 ) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 Å. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P i into mitochondria, the synthesis of ATP on F 1 , and the release and exit of ATP are very localized and highly coordinated events.Animal mitochondria have two major roles, one of which is to participate in cell life by making ATP by a process known as oxidative phosphorylation (1), and the other role is to participate in cell death, either by apoptosis or necrosis (2, 3). During oxidative phosphorylation the ATP synthase works in the direction of ATP synthesis, whereas during necrotic cell death the enzyme works in the direction of ATP hydrolysis (3, 4). When working in the direction of ATP synthesis in intact mitochondria, the enzyme has an absolute dependence on the P i and ADP/ATP carriers, PIC 1 and ANC, respectively (5). These carriers supply the ATP synthase with its essential substrates to make ATP, and ANC also transports ATP out of the mitochondria to energize numerous cellular processes (Fig. 1A).The ATP synthase has attracted considerable attention in recent years, not only because of its involvement in cell life and necrotic cell death, but also because of the unique and remarkable mechanism by which it makes ATP (6 -10). Although a consensus has not been reached on all points, it seems clear that the synthase consists of two nanomotors (11), one called F 1 , which is driven by ATP hydrolysis, and the other contained within F 0 , which is driven by a proton gradient. During oxidative phosphorylation in mitochondria (Fig. 1A), the electron transport chain generates a proton gradient that is purported to dr...

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Section: Discussionmentioning

confidence: 76%

Mitochondrial ATP Synthasome

Chen

Ko²,

Delannoy

et al. 2004

Journal of Biological Chemistry

190

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“…In addition to those transport complexes made up of subunits with four or six TM domains (7,8,33,34), larger proteins, such as glutamate (11) and glucose transporters (30), are also believed to be oligomeric. This property may be shared by all members of the Na ϩ -and Cl Ϫ -coupled transporter family or it may be unique to SERT.…”

Section: Discussionmentioning

confidence: 99%

Oligomerization of serotonin transporter and its functional consequences

Kilic¹

2000

Proceedings of the National Academy of Sciences

101

107

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Two forms of serotonin transporter (SERT) were prepared with different epitope tags. When co-expressed in HeLa cells, the form containing a FLAG tag (Res-FLAG) was associated with the form containing a c-myc tag (Sens-myc). Antibody against c-myc precipitated Res-FLAG from detergent extracts of cells expressing both forms, but not when Res-FLAG was expressed alone. The specificity of the interaction was demonstrated by the observation that anti-myc antibodies did not precipitate the unrelated vesicular stomatitis virus coat glycoprotein when it was co-expressed with Sens-myc. Sens-myc contained a reactive cysteine at position 172, which reacted with both (2-aminoethyl)methanethiosulfonate and N-biotinylaminoethyl methanethiosulfonate on the surface of intact cells. Sens-myc, but not Res-FLAG, was inactivated by these reagents. When co-expressed with Sens-myc, functionally active Res-FLAG was precipitated by immobilized streptavidin from digitonin-solubilized cells that had been treated with N-biotinylaminoethyl methanethiosulfonate. In cells co-expressing mixtures of Sens-myc and Res-FLAG, the amount of inactivation by (2-aminoethyl)methanethiosulfonate was less than expected if the two forms were independent. The results are consistent with a dimeric form of SERT with functional interactions between subunits, and with association of dimers into a higher order complex, possibly a tetramer. Serotonin transporter (SERT) is responsible for the accumulation of serotonin by neurons and peripheral cells. It is a target for antidepressant drugs and also for psychostimulants such as cocaine and amphetamine derivatives. SERT is part of a larger family of Na ϩ -and Cl Ϫ -dependent solute carriers including transporters for the biogenic amine neurotransmitters norepinephrine and dopamine, and for the amino acid transmitters ␥-aminobutyric acid and glycine, as well as other substrates. SERT is a polytopic membrane protein predicted to contain 12 transmembrane (TM) domains. The high degree of sequence identity between members of this gene family suggests that the structures of all these proteins are very similar.As transport proteins, carriers like SERT share many structural features with channels. One of the characteristics that distinguishes carriers from channels is that, in channels, the pore through which ions flow is believed to be formed by multiple similar subunits or domains, each of which donates a TM domain to form the channel (1). In contrast, carriers are believed to exist in two distinct states that bind internal and external substrate, respectively, and that are interconverted by conformational changes (2). These carriers contain approximately 12 TM domains that are believed to form the binding sites and the permeation pathway within each monomer. Judging by the size of particles in freeze-fracture images, Eskandari et al. (3) estimated that Na ϩ -glucose symporter was a monomer in the membrane. In lac permease of Escherichia coli, interaction between monomers was not observed, even when the monomers were co...

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“…the heterodimer) and, therefore, in a single b 0,ϩ AT catalytic subunit. Interestingly, mitochondrial carriers have a similar transport mechanism, although each transport pathway resides in a single six-transmembrane domain subunit of the functional dimeric carrier (47,48). Clearly, studies with purified and reconstituted HAT will be necessary to integrate the transport mechanism, the functional unit, and the structure of these transporters in a unique model.…”

Section: Discussionmentioning

confidence: 99%