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  • 1
    Online Resource
    Online Resource
    Insight into the function of a unique voltage-sensor protein (TMEM266) and its short form in mouse cerebellum
    Portland Press Ltd. ; 2022
    In:  Biochemical Journal Vol. 479, No. 11 ( 2022-06-17), p. 1127-1145
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 479, No. 11 ( 2022-06-17), p. 1127-1145
    Abstract: Voltage-sensing proteins generally consist of voltage-sensor domains and pore-gate domains, forming the voltage-gated ion channels. However, there are several unconventional voltage-sensor proteins that lack pore-gate domains, conferring them unique voltage-sensing machinery. TMEM266, which is expressed in cerebellum granule cells, is one of the interesting voltage-sensing proteins that has a putative intracellular coiled-coil and a functionally unidentified cytosolic region instead of a pore-gate domain. Here, we approached the molecular function of TMEM266 by performing co-immunoprecipitation experiments. We unexpectedly discovered that TMEM266 proteins natively interact with the novel short form splice variants that only have voltage-sensor domains and putative cytosolic coiled-coil region in cerebellum. The crystal structure of coiled-coil region of TMEM266 suggested that these coiled-coil regions play significant roles in forming homodimers. In vitro expression experiments supported the idea that short form TMEM266 (sTMEM266) or full length TMEM266 (fTMEM266) form homodimers. We also performed proximity labeling mass spectrometry analysis for fTMEM266 and sTMEM266 using Neuro-2A, neuroblastoma cells, and fTMEM266 showed more interacting molecules than sTMEM266, suggesting that the C-terminal cytosolic region in fTMEM266 binds to various targets. Finally, TMEM266-deficient animals showed the moderate abnormality in open-field test. The present study provides clues about the novel voltage-sensing mechanism mediated by TMEM266.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BCJ20220033
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2022
    detail.hit.zdb_id: 1473095-9
    SSG: 12
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  • 2
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    Insight into the mechanism of H + -coupled nucleobase transport
    Proceedings of the National Academy of Sciences ; 2023
    In:  Proceedings of the National Academy of Sciences Vol. 120, No. 33 ( 2023-08-15)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 120, No. 33 ( 2023-08-15)
    Abstract: Members of the nucleobase/ascorbic acid transporter (NAT) gene family are found in all kingdoms of life. In mammals, the concentrative uptake of ascorbic acid (vitamin C) by members of the NAT family is driven by the Na + gradient, while the uptake of nucleobases in bacteria is powered by the H + gradient. Here, we report the structure and function of PurT Cp , a NAT family member from Colwellia psychrerythraea . The structure of PurT Cp was determined to 2.80 Å resolution by X-ray crystallography. PurT Cp forms a homodimer, and each protomer has 14 transmembrane segments folded into a transport domain (core domain) and a scaffold domain (gate domain). A purine base is present in the structure and defines the location of the substrate binding site. Functional studies reveal that PurT Cp transports purines but not pyrimidines and that purine binding and transport is dependent on the pH. Mutation of a conserved aspartate residue close to the substrate binding site reveals the critical role of this residue in H + -dependent transport of purines. Comparison of the PurT Cp structure with transporters of the same structural fold suggests that rigid-body motions of the substrate-binding domain are central for substrate translocation across the membrane.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.2302799120
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2023
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 3
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    Online Resource
    Membrane transport proteins in melanosomes: Regulation of ions for pigmentation
    Elsevier BV ; 2020
    In:  Biochimica et Biophysica Acta (BBA) - Biomembranes Vol. 1862, No. 12 ( 2020-12), p. 183318-
    Details
    In: Biochimica et Biophysica Acta (BBA) - Biomembranes, Elsevier BV, Vol. 1862, No. 12 ( 2020-12), p. 183318-
    Type of Medium: Online Resource
    ISSN: 0005-2736
    URL: Article
    DOI: 10.1016/j.bbamem.2020.183318
    RVK:
    WA 15000
    Language: English
    Publisher: Elsevier BV
    Publication Date: 2020
    detail.hit.zdb_id: 2209384-9
    SSG: 12
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  • 4
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    Consensus mutagenesis approach improves the thermal stability of system x c − transporter, xCT , and enables cryo‐EM analyses
    Wiley ; 2020
    In:  Protein Science Vol. 29, No. 12 ( 2020-12), p. 2398-2407
    Details
    In: Protein Science, Wiley, Vol. 29, No. 12 ( 2020-12), p. 2398-2407
    Abstract: System x c − is an amino acid antiporter that imports L‐cystine into cells and exports intracellular L‐glutamate, at a 1:1 ratio. As L‐cystine is an essential precursor for glutathione synthesis, system x c − supports tumor cell growth through glutathione‐based oxidative stress resistance and is considered as a potential therapeutic target for cancer treatment. System x c − consists of two subunits, the light chain subunit SLC7A11 (xCT) and the heavy chain subunit SLC3A2 (also known as CD98hc or 4F2hc), which are linked by a conserved disulfide bridge. Although the recent structures of another SLC7 member, L‐type amino acid transporter 1 (LAT1) in complex with CD98hc, have provided the structural basis toward understanding the amino acid transport mechanism, the detailed molecular mechanism of xCT remains unknown. To revealthe molecular mechanism, we performed single‐particle analyses of the xCT‐CD98hc complex. As wild‐type xCT‐CD98hc displayed poor stability and could not be purified to homogeneity, we applied a consensus mutagenesis approach to xCT. The consensus mutated construct exhibited increased stability as compared to the wild‐type, and enabled the cryoelectron microscopy (cryo‐EM) map to be obtained at 6.2 Å resolution by single‐particle analysis. The cryo‐EM map revealed sufficient electron density to assign secondary structures. In the xCT structure, the hash and arm domains are well resolved, whereas the bundle domain shows some flexibility. CD98hc is positioned next to the xCT transmembrane domain. This study provides the structural basis of xCT, and our consensus‐based strategy could represent a good choice toward solving unstable protein structures.
    Type of Medium: Online Resource
    ISSN: 0961-8368 , 1469-896X
    URL: Issue
    URL: Article
    DOI: 10.1002/pro.v29.12
    DOI: 10.1002/pro.3966
    RVK:
    WA 15000
    Language: English
    Publisher: Wiley
    Publication Date: 2020
    detail.hit.zdb_id: 2000025-X
    SSG: 12
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  • 5
    Online Resource
    Online Resource
    A novel role of the C-terminus of b0,+AT in the ER–Golgi trafficking of the rBAT–b0,+AT heterodimeric amino acid transporter
    Portland Press Ltd. ; 2009
    In:  Biochemical Journal Vol. 417, No. 2 ( 2009-01-15), p. 441-448
    Details
    In: Biochemical Journal, Portland Press Ltd., Vol. 417, No. 2 ( 2009-01-15), p. 441-448
    Abstract: The heterodimeric complex composed of rBAT (related to b0,+ amino acid transporter), a single-membrane-spanning glycosylated heavy chain, and b0,+AT, a putative 12-membrane-spanning non-glycosylated light chain, is an amino acid transporter that mediates the activity of system b0,+, a major apical transport system for cystine and dibasic amino acids in renal proximal tubule and small intestine. The C-terminus of b0,+AT has been proposed to play an important role in the functional expression of the heterodimeric transporters. In the present study, to reveal the roles of the C-terminus, we analysed b0,+AT mutants whose C-termini were sequentially deleted or replaced by site-directed mutagenesis in polarized MDCKII (Madin–Darby canine kidney II), non-polarized HEK-293 (human embryonic kidney-293) and HeLa cells. Although the deletion of C-terminus of b0,+AT did not affect the formation of a heterodimer with rBAT, it resulted in the loss of apparent transport function, owing to the failure of the plasma-membrane targeting of rBAT–b0,+AT heterodimeric complex associated with incomplete glycosylation of rBAT. A motif-like sequence Val480-Pro481-Pro482 was identified in the C-terminus of b0,+AT to be responsible for the C-terminus action in promoting the trafficking of rBAT–b0,+AT heterodimeric complex from the ER (endoplasmic reticulum) to Golgi apparatus. This is, to our knowledge, the first demonstration of the active contribution of the C-terminus of a light-chain subunit to the intracellular trafficking of heterodimeric transporters. Because the motif-like sequence Val480-Pro481-Pro482 is well conserved among the C-termini of light-chain subunits, common regulatory mechanisms could be proposed among heterodimeric amino acid transporters.
    Type of Medium: Online Resource
    ISSN: 0264-6021 , 1470-8728
    URL: Article
    DOI: 10.1042/BJ20081798
    RVK:
    WA 15000
    Language: English
    Publisher: Portland Press Ltd.
    Publication Date: 2009
    detail.hit.zdb_id: 1473095-9
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  • 6
    Online Resource
    Online Resource
    Novel cystine transporter in renal proximal tubule identified as a missing partner of cystinuria-related plasma membrane protein rBAT/SLC3A1
    Proceedings of the National Academy of Sciences ; 2016
    In:  Proceedings of the National Academy of Sciences Vol. 113, No. 3 ( 2016-01-19), p. 775-780
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 113, No. 3 ( 2016-01-19), p. 775-780
    Abstract: Heterodimeric amino acid transporters play crucial roles in epithelial transport, as well as in cellular nutrition. Among them, the heterodimer of a membrane protein b 0,+ AT/SLC7A9 and its auxiliary subunit rBAT/SLC3A1 is responsible for cystine reabsorption in renal proximal tubules. The mutations in either subunit cause cystinuria, an inherited amino aciduria with impaired renal reabsorption of cystine and dibasic amino acids. However, an unsolved paradox is that rBAT is highly expressed in the S3 segment, the late proximal tubules, whereas b 0,+ AT expression is highest in the S1 segment, the early proximal tubules, so that the presence of an unknown partner of rBAT in the S3 segment has been proposed. In this study, by means of coimmunoprecipitation followed by mass spectrometry, we have found that a membrane protein AGT1/SLC7A13 is the second partner of rBAT. AGT1 is localized in the apical membrane of the S3 segment, where it forms a heterodimer with rBAT. Depletion of rBAT in mice eliminates the expression of AGT1 in the renal apical membrane. We have reconstituted the purified AGT1-rBAT heterodimer into proteoliposomes and showed that AGT1 transports cystine, aspartate, and glutamate. In the apical membrane of the S3 segment, AGT1 is suggested to locate itself in close proximity to sodium-dependent acidic amino acid transporter EAAC1 for efficient functional coupling. EAAC1 is proposed to take up aspartate and glutamate released into luminal fluid by AGT1 due to its countertransport so that preventing the urinary loss of aspartate and glutamate. Taken all together, AGT1 is the long-postulated second cystine transporter in the S3 segment of proximal tubules and a possible candidate to be involved in isolated cystinuria.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1519959113
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2016
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
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  • 7
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    The LeuT-fold neurotransmitter:sodium symporter MhsT has two substrate sites
    Proceedings of the National Academy of Sciences ; 2018
    In:  Proceedings of the National Academy of Sciences Vol. 115, No. 34 ( 2018-08-21)
    Details
    In: Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 115, No. 34 ( 2018-08-21)
    Abstract: Crystal structures of the neurotransmitter:sodium symporter MhsT revealed occluded inward-facing states with one substrate (Trp) bound in the primary substrate (S1) site and a collapsed extracellular vestibule, which in LeuT contains the second substrate (S2) site. In n -dodecyl-β- d -maltoside, the detergent used to prepare MhsT for crystallization, the substrate-to-protein binding stoichiometry was determined by using scintillation proximity to be 1 Trp:MhsT. Here, using the same experimental approach, as well as equilibrium dialysis, we report that in n -decyl-β- d -maltoside, or after reconstitution in lipid, MhsT, like LeuT, can simultaneously bind two Trp substrate molecules. Trp binding to the S2 site sterically blocks access to a substituted Cys at position 33 in the S2 site, as well as access to the deeper S1 site. Mutation of either the S1 or S2 site disrupts transport, consistent with previous studies in LeuT showing that substrate binding to the S2 site is an essential component of the transport mechanism.
    Type of Medium: Online Resource
    ISSN: 0027-8424 , 1091-6490
    URL: Article
    DOI: 10.1073/pnas.1717444115
    RVK:
    TA 1000
    RVK:
    WA 15000
    Language: English
    Publisher: Proceedings of the National Academy of Sciences
    Publication Date: 2018
    detail.hit.zdb_id: 209104-5
    detail.hit.zdb_id: 1461794-8
    SSG: 11
    SSG: 12
    Location Call Number Limitation Availability
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