Single Replacement Constructs of All Hydroxyl, Basic, and Acidic Amino Acids Identify New Function and Structure-Sensitive Regions of the Mitochondrial Phosphate Transport Protein

Wohlrab, Hartmut; Annese, Vincent; Haefele, Amanda

doi:10.1021/bi0117551

Cited by 23 publications

(26 citation statements)

References 17 publications

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“…Another possible site for proton coupling is the asymmetric H33. The equivalent residues in the ortholog ScMir1p are essential for function (18).…”

Section: Resultsmentioning

confidence: 99%

“…The mutations E95Q, K98A, E192Q, S195A, and K295A of the phosphate transporter ScMir1p (Fig. S10 in SI Appendix) reduced the transport activity to 72%, 3%, 39%, 3%, and 33% of the wild-type activity, showing that apolar substitutions render the transporter dysfunctional, whereas polar substitutions are less severe (18). Notably, mutations need not have a strong effect, because they may only change the transport mode from exchanger to uniporter or vice versa (see below).…”

Section: Discussionmentioning

confidence: 99%

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The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Robinson

Overy²,

Kunji³

2008

Proc. Natl. Acad. Sci. U.S.A.

200

363

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The structures of mitochondrial transporters and uncoupling proteins are 3-fold pseudosymmetrical, but their substrates and coupling ions are not. Thus, deviations from symmetry are to be expected in the substrate and ion-binding sites in the central aqueous cavity. By analyzing the 3-fold pseudosymmetrical repeats from which their sequences are made, conserved asymmetric residues were found to cluster in a region of the central cavity identified previously as the common substrate-binding site. Conserved symmetrical residues required for the transport mechanism were found at the water-membrane interfaces, and they include the three PX T he members of the mitochondrial transporter or carrier family translocate nucleotides, amino acids, inorganic ions, keto acids, and vitamins across the mitochondrial inner membrane (1, 2). Uncoupling proteins, which generate heat by dissipating the proton electrochemical gradient, also belong to the family (3-5). The amino acid sequences have 3 homologous repeats (6) and a structure with pseudosymmetry (7). Each repeat is folded into 2 transmembrane ␣-helices linked by a short ␣-helix on the matrix side (8) and contains the signature motif PX[DE]XX[RK] (9). The proline residues kink the oddnumbered transmembrane ␣-helices H1, H3, and H5, and the charged residues form a salt-bridge network connecting the C-terminal end of the transmembrane ␣-helices, closing the transporter on the matrix side (8). During the transport cycle, the carriers form the cytoplasmic and matrix states in which the substrate-binding site of the carrier is open to the mitochondrial intermembrane space and matrix, respectively (10). According to the single binding center-gating pore mechanism, interconversion of the 2 conformational states via a transition intermediate leads to substrate translocation (11). In agreement, in the cytoplasmic state, a central substrate-binding site has been identified by applying chemical and distance constraints to comparative models (12, 13). The substrates bind to 3 major sites on the even-numbered ␣-helices, which are related by symmetry and located approximately in the middle of the membrane. In molecular dynamics simulations, ADP binds to the ADP/ATP carrier at the common substrate-binding site (14). The structural changes required for the translocation of the substrate are unknown, but docking of the substrate could disrupt the matrix salt bridge network, allowing the transporter to convert to the matrix state (12, 13). The yeast ADP/ATP carriers function as monomers (15), and other mitochondrial transporters are likely to operate in the same way.Residues that are important for the transport mechanism are likely to be symmetrical, whereas residues involved in substrate binding will be asymmetrical reflecting the asymmetry of the substrates. By scoring the symmetry of residues in the sequence repeats, we have identified the substrate-binding sites and salt bridge networks that are important for the transport mechanism in family members. No preexisting molecular structure i...

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“…Another possible site for proton coupling is the asymmetric H33. The equivalent residues in the ortholog ScMir1p are essential for function (18).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Robinson

Overy²,

Kunji³

2008

Proc. Natl. Acad. Sci. U.S.A.

200

363

View full text Add to dashboard Cite

show abstract

“…The phosphate carrier Mir1p has basic residues at contact points I and II that mutagenesis shows are important for its transport function (15). The binding site is similar to that of keto acid carriers, but a single phosphate anion is too small to be bound at contact points I and II simultaneously.…”

Section: Resultsmentioning

confidence: 99%

“…Mutation of residue 182 BtAAC1 at contact point II affect carrier function in yeast Mir1p (15) and Ctp1p (18), human OGC (19) and UCP1 (20), and hamster mitochondrial folate transporter͞carrier (12). Hyperornithinemia-hyperammonia-homocitrullinuria syndrome is associated with the mutation E180K in ornithine transporter (ORNT) 1 (21).…”

Section: Resultsmentioning

confidence: 99%

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Robinson

Kunji

2006

Proc. Natl. Acad. Sci. U.S.A.

237

289

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Mitochondrial carriers link biochemical pathways in the cytosol and mitochondrial matrix by transporting substrates across the inner mitochondrial membrane. Substrate recognition is specific for each carrier, but sequence similarities suggest the carriers have similar structures and mechanisms of substrate translocation. By considering conservation of amino acids, distance and chemical constraints, and by modeling family members on the known structure of the ADP͞ATP translocase, we have identified a common substrate binding site. It explains substrate selectivity and proton coupling and provides a mechanistic link to carrier opening by substrate-induced perturbation of the salt bridges that seal the pathway to and from the mitochondrial matrix. It enables the substrate specificity of uncharacterized mitochondrial carriers to be predicted.comparative modeling ͉ membrane protein ͉ sequence alignment M embers of the mitochondrial carrier family are located in the inner mitochondrial membrane and allow exchange of substrates between the cytosol and the mitochondrial matrix (1). The family is exclusive to eukaryotes, and its members are unrelated to other transporter families, including the major facilitator superfamily and the ABC transporters. Their substrates include nucleotides, amino acids, cofactors, carboxylic acids, and inorganic anions required by the organelle for oxidative phosphorylation, gluconeogenesis, synthesis and degradation of amino and fatty acids, macromolecular synthesis of proteins and nucleic acids, sterol metabolism, and thermogenesis (1). Mutations of the carriers are associated with diseases, including progressive external opthalmoplegia, adult-and neonatal-onset type II citrullinaemia, Amish-type microcephaly, hyperornithinemia-hyperammonia-homocitrullinuria, carnitineacylcarnitine translocase deficiency, neonatal myoclonic epilepsy, severe obesity, and type II diabetes (1).The mitochondrial carriers have three tandem repeats of Ϸ100 aa, each containing two transmembrane ␣-helices linked by a large loop (2) and a conserved signature motif P-X-[DE]-X-X-[RK] (3). The carriers exist in two distinct conformational states: the cytoplasmic state (c-state) in which the carrier accepts its substrate from the cytoplasm, and the matrix state in which it accepts a substrate from the mitochondrial matrix. For the ADP͞ATP carrier, the inhibitor carboxyatractyloside (CATR) is known to bind to the carrier in the c-state only and prevent the binding of ADP. In the atomic model of the ADP͞ATP carrier 1 from Bos taurus (BtAAC1) in complex with CATR (4), the six-transmembrane ␣-helices form a barrel that has pseudo-3-fold symmetry. CATR is bound in the internal aqueous cavity (see Fig. 4, which is published as supporting information on the PNAS web site), which is open to the cytosol and closed to the mitochondrial matrix, consistent with the structure representing the c-state. The prolines of the signature motif kink the transmembrane helices, bringing their C-terminal ends together at the base of the cavit...

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“…To overcome this dilemma, the heterologous expression/reconstitution system, as discussed already, is indeed most helpful. Using this approach, a most detailed analysis has been carried out with the phosphate transporter (32). These studies led to likely phosphate and proton transport paths within the protein and identified two amino acids that may be central in the synchronization of these two pathways to yield the physiologically relevant electroneutral and proton-compensated phosphate transport (Figs.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

Transport proteins (carriers) of mitochondria

Wohlrab

2008

IUBMB Life

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SummaryMitochondria are subcellular structures essential to the aerobic eukaryotic cell. Their role extends much beyond their basic reactions of oxidative phosphorylation. It encompasses the steps critical for cellular metabolic pathways, for apoptosis, and for other processes such as antiviral signaling. This short review is limited to transport proteins (carriers) that catalyze the transport of metabolites across the inner mitochondrial membrane and thus link metabolic pathway reactions in the cytosol and the mitochondrial matrix. Such transport must minimally affect the electrochemical proton gradient essential for oxidative phosphorylation (chemiosmotic mechanism of oxidative phosphorylation). Many of these transport proteins belong to a family of membrane proteins, and the major part of this review will consider their structures and functions. First studies of these transporters were carried out with intact mitochondria and with inhibitors that appeared transporter-specific. Such an inhibitor was then utilized in the first purification of one of these transporter proteins. Its substrate-specificity was then established after functionally active incorporation into liposomes. Questions about copurification of other transporters and thus a definitive identification of transported substrate with the purified protein were resolved definitively only after heterologous expression in bacteria, most generally as inclusion bodies, and followed by reconstitution in liposomes. Site-specific mutations permitted the identification of amino acids essential to their transport function. These mutagenesis studies then also helped interpret human diseases with mutations in these transport proteins. The high-resolution structure of a member of this transporter protein family dramatically advanced these studies. It raised new questions because this structure complexed with a high-affinity inhibitor showed a monomeric protein, while purification and inhibitor stoichiometry studies suggest a functional homodimeric transport protein. Remaining key questions need to address: the homodimeric nature of the transporters, details of their transport mechanism, and the functional identification of many members of this family whose existence has only been suggested from genomic data. IUBMBIUBMB Life, 61(1): [40][41][42][43][44][45][46] 2009

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Single Replacement Constructs of All Hydroxyl, Basic, and Acidic Amino Acids Identify New Function and Structure-Sensitive Regions of the Mitochondrial Phosphate Transport Protein

Cited by 23 publications

References 17 publications

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

The mechanism of transport by mitochondrial carriers based on analysis of symmetry

Mitochondrial carriers in the cytoplasmic state have a common substrate binding site

Transport proteins (carriers) of mitochondria

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