Publication

PnuT uses a facilitated diffusion mechanism for thiamine uptake

Jaehme, M., Singh, R., Garaeva, A. A., Duurkens, R. H. & Slotboom, D-J., Dec-2017, In : Journal of general physiology. 150, 1, p. 41-50 9 p., jgp.201711850.

Research output: Contribution to journalArticleAcademicpeer-review

APA

Jaehme, M., Singh, R., Garaeva, A. A., Duurkens, R. H., & Slotboom, D-J. (2017). PnuT uses a facilitated diffusion mechanism for thiamine uptake. Journal of general physiology, 150(1), 41-50. [jgp.201711850]. https://doi.org/10.1085/jgp.201711850

Author

Jaehme, Michael ; Singh, Rajkumar ; Garaeva, Alisa A ; Duurkens, Ria H ; Slotboom, Dirk-Jan. / PnuT uses a facilitated diffusion mechanism for thiamine uptake. In: Journal of general physiology. 2017 ; Vol. 150, No. 1. pp. 41-50.

Harvard

Jaehme, M, Singh, R, Garaeva, AA, Duurkens, RH & Slotboom, D-J 2017, 'PnuT uses a facilitated diffusion mechanism for thiamine uptake' Journal of general physiology, vol. 150, no. 1, jgp.201711850, pp. 41-50. https://doi.org/10.1085/jgp.201711850

Standard

PnuT uses a facilitated diffusion mechanism for thiamine uptake. / Jaehme, Michael; Singh, Rajkumar; Garaeva, Alisa A; Duurkens, Ria H; Slotboom, Dirk-Jan.

In: Journal of general physiology, Vol. 150, No. 1, jgp.201711850, 12.2017, p. 41-50.

Research output: Contribution to journalArticleAcademicpeer-review

Vancouver

Jaehme M, Singh R, Garaeva AA, Duurkens RH, Slotboom D-J. PnuT uses a facilitated diffusion mechanism for thiamine uptake. Journal of general physiology. 2017 Dec;150(1):41-50. jgp.201711850. https://doi.org/10.1085/jgp.201711850


BibTeX

@article{aacb2738405f4839b49f8d3529716533,
title = "PnuT uses a facilitated diffusion mechanism for thiamine uptake",
abstract = "Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.",
keywords = "COUPLING FACTOR TRANSPORTER, MEMBRANE-PROTEINS, CRYSTAL-STRUCTURE, ABC TRANSPORTERS, ECF TRANSPORTER, BINDING, IDENTIFICATION, BIOSYNTHESIS, PROKARYOTES, BACTERIA",
author = "Michael Jaehme and Rajkumar Singh and Garaeva, {Alisa A} and Duurkens, {Ria H} and Dirk-Jan Slotboom",
note = "{\circledC} 2018 Jaehme et al.",
year = "2017",
month = "12",
doi = "10.1085/jgp.201711850",
language = "English",
volume = "150",
pages = "41--50",
journal = "Journal of general physiology",
issn = "0022-1295",
number = "1",

}

RIS

TY - JOUR

T1 - PnuT uses a facilitated diffusion mechanism for thiamine uptake

AU - Jaehme, Michael

AU - Singh, Rajkumar

AU - Garaeva, Alisa A

AU - Duurkens, Ria H

AU - Slotboom, Dirk-Jan

N1 - © 2018 Jaehme et al.

PY - 2017/12

Y1 - 2017/12

N2 - Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.

AB - Membrane transporters of the bacterial pyridine nucleotide uptake (Pnu) family mediate the uptake of various B-type vitamins. For example, the PnuT transporters have specificity for vitamin B1 (thiamine). It has been hypothesized that Pnu transporters are facilitators that allow passive transport of the vitamin substrate across the membrane. Metabolic trapping by phosphorylation would then lead to accumulation of the transported substrates in the cytoplasm. However, experimental evidence for such a transport mechanism is lacking. Here, to determine the mechanism of thiamine transport, we purify PnuTSw from Shewanella woodyi and reconstitute it in liposomes to determine substrate binding and transport properties. We show that the electrochemical gradient of thiamine solely determines the direction of transport, consistent with a facilitated diffusion mechanism. Further, PnuTSw can bind and transport thiamine as well as the thiamine analogues pyrithiamine and oxythiamine, but does not recognize the phosphorylated derivatives thiamine monophosphate and thiamine pyrophosphate as substrates, consistent with a metabolic trapping mechanism. Guided by the crystal structure of the homologous nicotinamide riboside transporter PnuC, we perform mutagenesis experiments, which reveal residues involved in substrate binding and gating. The facilitated diffusion mechanism of transport used by PnuTSw contrasts sharply with the active transport mechanisms used by other bacterial thiamine transporters.

KW - COUPLING FACTOR TRANSPORTER

KW - MEMBRANE-PROTEINS

KW - CRYSTAL-STRUCTURE

KW - ABC TRANSPORTERS

KW - ECF TRANSPORTER

KW - BINDING

KW - IDENTIFICATION

KW - BIOSYNTHESIS

KW - PROKARYOTES

KW - BACTERIA

U2 - 10.1085/jgp.201711850

DO - 10.1085/jgp.201711850

M3 - Article

VL - 150

SP - 41

EP - 50

JO - Journal of general physiology

JF - Journal of general physiology

SN - 0022-1295

IS - 1

M1 - jgp.201711850

ER -

ID: 51452526