Cryo-EM structure of the human heteromeric amino acid transporter b
            <sup>0,+</sup>
            AT-rBAT

Yan, Renhong; Li, Yaning; Shi, Yi; Zhou, Jiayao; Lei, Jianlin; Huang, Jing; Zhou, Qiang

doi:10.1126/sciadv.aay6379

Cited by 37 publications

(67 citation statements)

References 62 publications

(69 reference statements)

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“…1d-e). This architecture is essentially identical to the recent structures of human b 0,+ AT-rBAT (14), confirming a conserved higher-order assembly (Fig. S6b).…”

Section: Overall Structure Of the Ovine B 0+ At-rbat Complexsupporting

confidence: 83%

“…Single-particle analysis indicated that the two b 0,+ AT-rBAT subcomplexes are linked together with substantial flexibility, which was also evident in the human complex that showed a blurred TMD map in the consensus 3D refinement (14,15). To account for this flexibility, we performed multi-body refinement (16), taking individual heterodimers as rigid bodies moving relative to each other.…”

Section: The B 0+ At-rbat Interfacementioning

confidence: 99%

“…In addition, while all characterized type I mutations show the disease phenotype only in homozygotes, some non-type I mutations can trigger the disease even in heterozygous individuals (13), a phenomenon that currently lacks a clear explanation. Recently, the cryo-EM structures of human b 0,+ AT-rBAT in detergent revealed its architecture and suggested a mechanism of amino acid antiport and its dysfunctions (14,15). However, these structures did not explain why certain type I mutations cause protein trafficking defects.…”

Section: Introductionmentioning

confidence: 99%

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Ca²⁺-mediated higher-order assembly of b^0,+AT–rBAT is a key step for system b^0,+biogenesis and cystinuria

Lee

Wiriyasermkul

Moriyama

et al. 2021

Preprint

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Cystinuria is a genetic disorder characterized by overexcretion of dibasic amino acids and cystine, which causes recurrent kidney stones and occasionally severe kidney failure. Mutations of the two responsible proteins, rBAT and b0,+AT, which comprise system b0,+, are linked to type I and non-type I cystinuria respectively and they exhibit distinct phenotypes due to protein trafficking defects or catalytic inactivation. Although recent structural insights into human b0,+AT-rBAT suggested a model for transport-inactivating mutations, the mechanisms by which type I mutations trigger trafficking deficiencies are not well understood. Here, using electron cryo-microscopy and biochemistry, we discover that Ca2+-mediated higher-order assembly of system b0,+ is the key to its trafficking on the cell surface. We show that Ca2+ stabilizes the interface between two rBAT molecules to mediate super-dimerization, and this in turn facilitates the N-glycan maturation of system b0,+. A common cystinuria mutant T216M and mutations that disrupt the Ca2+ site in rBAT cause the loss of higher-order assemblies, resulting in protein trafficking deficiency. Mutations at the super-dimer interface reproduce the mis-trafficking phenotype, demonstrating that super-dimerization is essential for cellular function. Cell-based transport assays confirmed the importance of the Ca2+ site and super-dimerization, and additionally suggested which residues are involved in cationic amino acid recognition. Taken together, our results provide the molecular basis of type I cystinuria and serve as a guide to develop new therapeutic strategies against it. More broadly, our findings reveal an unprecedented link between transporter oligomeric assembly and trafficking diseases in general.

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“…1d-e). This architecture is essentially identical to the recent structures of human b 0,+ AT-rBAT (14), confirming a conserved higher-order assembly (Fig. S6b).…”

Section: Overall Structure Of the Ovine B 0+ At-rbat Complexsupporting

confidence: 83%

Section: The B 0+ At-rbat Interfacementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Ca²⁺-mediated higher-order assembly of b^0,+AT–rBAT is a key step for system b^0,+biogenesis and cystinuria

Lee

Wiriyasermkul

Moriyama

et al. 2021

Preprint

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“…Therefore, LAT1 is a potential drug target in cancer but it may also be exploited for drug delivery into the brain. The inward-open conformation cryo-EM structures of the human LAT1-4F2hc complex in its apo state or with the non-specific inhibitor 2-amino-2-norbornanecarboxylic acid (BCH) bound and some eukaryotic homologs of the HAT family were solved [12][13][14][15] . However, structures of LAT1 or other HAT family members in other conformations with or without inhibitors bound are not available yet.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

Yan

Müller

et al. 2021

Cell Discov

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LAT1 (SLC7A5) is one of the representative light chain proteins of heteromeric amino acid transporters, forming a heterodimer with its heavy chain partner 4F2hc (SLC3A2). LAT1 is overexpressed in many types of tumors and mediates the transfer of drugs and hormones across the blood-brain barrier. Thus, LAT1 is considered as a drug target for cancer treatment and may be exploited for drug delivery into the brain. Here, we synthesized three potent inhibitors of human LAT1, which inhibit transport of leucine with IC50 values between 100 and 250 nM, and solved the cryo-EM structures of the corresponding LAT1-4F2hc complexes with these inhibitors bound at resolution of up to 2.7 or 2.8 Å. The protein assumes an outward-facing occluded conformation, with the inhibitors bound in the classical substrate binding pocket, but with their tails wedged between the substrate binding site and TM10 of LAT1. We also solved the complex structure of LAT1-4F2hc with 3,5-diiodo-l-tyrosine (Diiodo-Tyr) at 3.4 Å overall resolution, which revealed a different inhibition mechanism and might represent an intermediate conformation between the outward-facing occluded state mentioned above and the outward-open state. To our knowledge, this is the first time that the outward-facing conformation is revealed for the HAT family. Our results unveil more important insights into the working mechanisms of HATs and provide a structural basis for future drug design.

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“…Cryo- and negative stain-electron microscopy (EM) elucidated the supramolecular organization and structures of selected HATs, i.e., of 4F2hc-LAT1 [ 22 , 23 , 24 ], 4F2hc-LAT2 [ 25 , 26 , 27 , 28 ], and rBAT-b 0,+ AT [ 29 ]. Furthermore, the high-resolution cryo-EM structures of 4F2hc-LAT1 [ 22 , 23 ] and rBAT-b 0,+ AT [ 29 ] provided detailed insights into the interactions between the ancillary glycoproteins and the corresponding membrane transporters at the molecular level.…”

Section: Introductionmentioning

confidence: 99%

The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

Kantipudi

Jeckelmann

Ucurum

et al. 2020

IJMS

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The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

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Cryo-EM structure of the human heteromeric amino acid transporter b ^0,+ AT-rBAT

Cited by 37 publications

References 62 publications

Ca²⁺-mediated higher-order assembly of b^0,+AT–rBAT is a key step for system b^0,+biogenesis and cystinuria

Ca²⁺-mediated higher-order assembly of b^0,+AT–rBAT is a key step for system b^0,+biogenesis and cystinuria

Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

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Cryo-EM structure of the human heteromeric amino acid transporter b 0,+ AT-rBAT

Cited by 37 publications

References 62 publications

Ca2+-mediated higher-order assembly of b0,+AT–rBAT is a key step for system b0,+biogenesis and cystinuria

Ca2+-mediated higher-order assembly of b0,+AT–rBAT is a key step for system b0,+biogenesis and cystinuria

Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

Contact Info

Product

Resources

About

Cryo-EM structure of the human heteromeric amino acid transporter b ^0,+ AT-rBAT

Ca²⁺-mediated higher-order assembly of b^0,+AT–rBAT is a key step for system b^0,+biogenesis and cystinuria

Ca²⁺-mediated higher-order assembly of b^0,+AT–rBAT is a key step for system b^0,+biogenesis and cystinuria