Transmembrane Cu(<scp>i</scp>) P-type ATPase pumps are electrogenic uniporters

Abeyrathna, Nisansala; Abeyrathna, Sameera; Morgan, M. Thomas; Fahrni, Christoph J.; Meloni, Gabriele

doi:10.1039/d0dt01380c

Cited by 16 publications

(21 citation statements)

References 63 publications

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“…It was concluded that the electrogenicity of ATP8A2 is related to a step in the ATPase transport cycle directly involved in translocation of the phospholipid [ 32 ]: dephosphorylation of the E 2 P intermediate, activated by lipid binding from the exoplasmic side, and/or the subsequent E 2 → E 1 conformational transition of the dephosphoenzyme, which is associated with release of the lipid to the cytoplasmic side [ 104 ]. It is noteworthy that the findings of the SSM study of the mammalian phospholipid flippase [ 32 ] support the view that the P4-ATPase is most likely an electrogenic uniporter, as also recently reported for a bacterial Cu + -transporting P1B-ATPase [ 82 ].…”

Section: P-type Atpases Investigated On Solid Supported Membranessupporting

confidence: 61%

“…Thus, SSM measurements on ATP7A/B supported the hypothesis that Cu + release in these enzymes may not be coupled to a net proton countertransport, which has not been observed for PIB-type ATPases [ 72 , 73 , 81 ]. Interestingly, a very recent study reported real-time fluorescence measurements on E.coli Cu + -ATPase (EcCopA) reconstituted in small unilamellar vesicles encapsulating a set of fluorescence probes that are selective for Cu + , pH, and membrane potential [ 82 ]. The results of this study demonstrated the absence of H + countertransport in the Cu + translocation cycle of EcCopA, qualifying EcCopA as an electrogenic uniporter.…”

Section: P-type Atpases Investigated On Solid Supported Membranesmentioning

confidence: 99%

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Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

Tadini‐Buoninsegni

2020

Molecules

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P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

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Section: P-type Atpases Investigated On Solid Supported Membranessupporting

confidence: 61%

Section: P-type Atpases Investigated On Solid Supported Membranesmentioning

confidence: 99%

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

Tadini‐Buoninsegni

2020

Molecules

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“…Cu transport by CopA-like P 1B -type ATPases has been extensively studied using different methods ( Gonzalez-Guerrero et al, 2010 ; Völlmecke et al, 2012 ; Drees et al, 2015 ; Abeyrathna et al, 2020 ; Tadini-Buoninsegni, 2020 ), but similar information on CcoI-type ATPases is limited. Cu transport by CopA1 and CopA2 of Pseudomonas aeruginosa has been determined by using E. coli expressed proteins and monitoring 64 Cu accumulation in membrane vesicles ( Gonzalez-Guerrero et al, 2010 ).…”

Section: Resultsmentioning

confidence: 99%

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

et al. 2021

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Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.

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“…45,46 These catalytically driven pumps constitute an essential system to drive the selective translocation and export of Cu + ions, thereby controlling the intracellular Cu + levels. 47,48 Their activity tightly balances the biogenesis and integrity of copper centers in vital enzymes to non-toxic intracellular copper levels. The CopA structure is characterized by the existence of an 8 TM helices membrane domain (M-domain) connected to large cytosolic domains (N-, P-and A-domains) responsible for ATP hydrolysis, phosphorylation and energy transduction, allowing Cu + translocation across the membrane.…”

Section: Stabilization Of Purified Transmembrane Proteinsmentioning

confidence: 99%

“…The CopA structure is characterized by the existence of an 8 TM helices membrane domain (M-domain) connected to large cytosolic domains (N-, P-and A-domains) responsible for ATP hydrolysis, phosphorylation and energy transduction, allowing Cu + translocation across the membrane. 35,45,47,49 As a result of their critical involvement in essential iron and copper metabolism, both IroT and CopA homologues have been identified as key virulence factors in bacterial pathogens. 37,50 Transmembrane proteins, including IroT and CopA, are commonly extracted from membranes and purified as detergent micellar complexes for solubilization in aqueous environments.…”

Section: Stabilization Of Purified Transmembrane Proteinsmentioning

confidence: 99%

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

Herbert¹,

Abeyrathna²,

Abeyrathna³

et al. 2021

Preprint

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<div> <div> <div> <p>Artificial native-like lipid bilayer systems constructed from phospholipids assembling into unilamel- lar liposomes allow the reconstitution of detergent-solubilized transmembrane proteins into supramolecular lipid-protein assemblies called proteoliposomes, which mimic cellular membranes. Stabilization of these com- plexes remains challenging because of their chemical composition, the hydrophobicity and structural instabil- ity of membrane proteins, and the lability of interactions between protein, detergent, and lipids within micelles and lipid bilayers. In this work we demonstrate that metastable lipid, protein-detergent, and protein-lipid su- pramolecular complexes can be successfully generated and immobilized within zeolitic-imidazole framework- 8 (ZIF-8) to enhance their stability against chemical and physical stressors. Upon immobilization in ZIF-8 bio- composites, blank liposomes, and model transmembrane metal transporters in detergent micelles or embed- ded in proteoliposomes resist elevated temperatures, exposure to chemical denaturants, aging, and mechanical stresses. Extensive morphological and functional characterization of the assemblies upon exfoliation reveal that all these complexes encapsulated within the framework maintain their native morphology, structure, and activity, which is otherwise lost rapidly without immobilization. </p> </div> </div> </div>

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Transmembrane Cu(i) P-type ATPase pumps are electrogenic uniporters

Abstract: Real-time transport analyses define transmembrane Cu(i)-pumps as electrogenic uniporters.

Cited by 16 publications

References 63 publications

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

Stabilization of Supramolecular Membrane Protein-Lipid Bilayer Assemblies Through Immobilization in a Crystalline Exoskeleton

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