Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Zheng, Lu; Lee, Ashley S.E.; Bracher, Susanne; Jung, Heinrich; Paz, Adrian; Kumar, J.; Abramson, Jeff; Quick, Matthias; Shi, Lei

doi:10.1074/jbc.m114.584383

Cited by 18 publications

(21 citation statements)

References 72 publications

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“…Of the crucial amino acids of TM6Ј, Trp-244, Tyr-248, and Pro-252 may participate in proline binding as already suggested previously (18,23). The idea agrees well with the central role of TM6Ј in substrate binding described for other transporters with a LeuT-type structural fold (8, 12, 14, 24 -26, 28).…”

Section: Substitutionsupporting

confidence: 89%

“…On the contrary, previous transport and proline binding analyses demonstrated that substitutions for Trp-244 (W244C or -F; 10 2 -10 3 -fold increase of K m(Pro) ) (18), Tyr-248 (Y248C, -F, or -T; up to 10-fold increase of K m(Pro) ) (18, 23), and Pro-252 (Cys) affect binding of proline to PutP (Ref. 23 and this investigation). The severely increased K m(Pro) of W244C (18) is consistent with the reduced binding signal observed here with the DRaCALA (Fig.…”

Section: Resultsmentioning

confidence: 93%

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Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP

Bracher

Schmidt

Dittmer

et al. 2016

Journal of Biological Chemistry

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Crystal structures of transporters with a LeuT-type structural fold assign core transmembrane domain 6 (TM6') a central role in substrate binding and translocation. Here, the function of TM6' in the sodium/proline symporter PutP, a member of the solute/sodium symporter family, was investigated. A complete scan of TM6' identified eight amino acids as particularly important for PutP function. Of these residues, Tyr-248, His-253, and Arg-257 impact sodium binding, whereas Arg-257 and Ala-260 may participate in interactions leading to closure of the inner gate. Furthermore, the previous suggestion of an involvement of Trp-244, Tyr-248, and Pro-252 in proline binding is further supported. In addition, substitution of Gly-245, Gly-247, and Gly-250 affects the amount of PutP in the membrane. A Cys accessibility analysis suggests an involvement of the inner half of TM6' in the formation of a hydrophilic pathway that is open to the inside in the absence of ligands and closed in the presence of sodium and proline. In conclusion, the results demonstrate that TM6' plays a central role in substrate binding and release on the inner side of the membrane also in PutP and extend the knowledge on functionally relevant amino acids in transporters with a LeuT-type structural fold.

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Section: Substitutionsupporting

confidence: 89%

Section: Resultsmentioning

confidence: 93%

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP

Bracher

Schmidt

Dittmer

et al. 2016

Journal of Biological Chemistry

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“…Previous work suggests that the LeuT, SERT, NET, and DAT transporters, to which SLC38A9 is distantly related, have two small molecule binding sites, one for the transported metabolite and another for a molecule that allosterically regulates transport (Li et al, 2015; Neubauer et al, 2006; Quick et al, 2012; Shi et al, 2008; Singh et al, 2007; Zhou et al, 2007). Our in vitro data suggest that arginine increases the V max of SLC38A9 for leucine transport without affecting its K m for leucine.…”

Section: Discussionmentioning

confidence: 99%

mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient

Wyant

Abu-Remaileh

Wolfson

et al. 2017

Cell

369

352

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Summary The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.

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“…Indeed, extensive computational modelling and simulations of the system [27, 32-38] focused on the detailed manner in which binding of substrate and ions from the extracellular side of the transporter induces conformational changes in the protein leading to the translocation of substrate and ions towards the intracellular vestibule. Furthermore, ligand binding and uptake measurements in LeuT together with computational modelling [32, 48, 49] have suggested a mechanistic model of allosteric Na + -coupled symport in which intracellular release of the S1-bound substrate is triggered by the binding of a second substrate molecule in the extracellular vestibule located ~11Å above the S1 site, termed the S2 site.…”

Section: Introductionmentioning

confidence: 99%

Functional mechanisms of neurotransmitter transporters regulated by lipid–protein interactions of their terminal loops

Khelashvili

Weinstein

2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP2) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP2 lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP2 lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins.

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Identification of a Second Substrate-binding Site in Solute-Sodium Symporters

Cited by 18 publications

References 72 publications

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP

Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP

mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient

Functional mechanisms of neurotransmitter transporters regulated by lipid–protein interactions of their terminal loops

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