Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

Pochini, Lorena; Galluccio, Michele

doi:10.3390/life12081206

Cited by 3 publications

(3 citation statements)

References 241 publications

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“…Finally, this work reiterates the difficulty of predicting the substrate specificity of transporters based just on sequence similarity and the need to combine comparative genomics with experimental validation to functionally annotate this functional group. Systematic efforts to screen transporter substrate specificity are being implemented [ 48 , 49 , 50 ]. These will need to be combined with specialized transporter annotation capture and propagation tools to generally improve the current transporter databases [ 46 , 51 ].…”

Section: Discussionmentioning

confidence: 99%

Deciphering the Diversity in Bacterial Transporters That Salvage Queuosine Precursors

Quaiyum,

Yuan,

Kuipers

et al. 2024

Epigenomes

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Queuosine (Q) is a modification of the wobble base of tRNA harboring GUN anticodons with roles in decoding accuracy and efficiency. Its synthesis is complex with multiple enzymatic steps, and several pathway intermediates can be salvaged. The only two transporter families known to salvage Q precursors are QPTR/COG1738 and QrtT/QueT. Analyses of the distribution of known Q synthesis and salvage genes in human gut and oral microbiota genomes have suggested that more transporter families remain to be found and that Q precursor exchanges must occur within the structured microenvironments of the mammalian host. Using physical clustering and fusion-based association with Q salvage genes, candidate genes for missing transporters were identified and five were tested experimentally by complementation assays in Escherichia coli. Three genes encoding transporters from three different Pfam families, a ureide permease (PF07168) from Acidobacteriota bacterium, a hemolysin III family protein (PF03006) from Bifidobacterium breve, and a Major Facilitator Superfamily protein (PF07690) from Bartonella henselae, were found to allow the transport of both preQ0 and preQ1 in this heterologous system. This work suggests that many transporter families can evolve to transport Q precursors, reinforcing the concept of transporter plasticity.

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Section: Discussionmentioning

confidence: 99%

Deciphering the Diversity in Bacterial Transporters That Salvage Queuosine Precursors

Quaiyum,

Yuan,

Kuipers

et al. 2024

Epigenomes

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show abstract

“…Finally, this work reiterates the difficulty of predicting the substrate specificity of transporters based just on sequence similarity and the need to combine comparative genomics with experimental validation to functionally annotate this functional group. Systematic efforts to screen transporter substrate specificity are being implemented (43)(44)(45). These will need to be combined with specialized transporter annotation capture and propagation tools to generally improve the current transporter databases (41,46).…”

Section: Discussionmentioning

confidence: 99%

Deciphering the Diversity of Bacterial Transporters that Salvage Queuosine Precursors

Quaiyum,

Yuan,

Kuipers

et al. 2024

Preprint

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Queuosine (Q) is a modification of the wobble base of tRNA harboring GUN antico-dons with roles in decoding accuracy and efficiency. Its synthesis is complex with mul-tiple enzymatic steps and several pathway intermediates can be salvaged. The only two transporter families known to salvage Q precursors are QPTR/COG1738 and QrtT/QueT. Analysis of the distribution of known Q synthesis and salvage genes in human gut and oral microbiota genomes have suggested that more transporter fami-lies remained to be found and that Q precursor exchanges must occur within the struc-tured microenvironments of the mammalian host. Using physical clustering and fu-sion-based association with Q salvage genes, candidate genes for missing transporters were identified and five were tested experimentally by complementation assays in Escherichia coli. Three genes encoding transporters from three different Pfam families, a Ureide permease (PF07168) from Acidobacteriota bacterium, a Hemolysin III family pro-tein (PF03006) from Bifidobacterium breve, and a Major Facilitator Superfamily protein (PF07690) from Bartonella henselae, were found to allow the transport of both preQ0 and preQ1 in this heterologous system. This work suggests that many transporter families can evolve to transport Q precursors reinforcing the concept of transporter plasticity.

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“…The methylotrophic yeast Pichia pastoris is a well-established cellular factory for the production of recombinant mammalian membrane proteins ( Byrne, 2015 ; Looser et al, 2015 ; Pochini and Galluccio, 2022 ). Over the years, different human LATs and HATs were successfully overexpressed in P. pastoris for functional and structural studies ( Costa et al, 2013 ; Meury et al, 2014 ; Rosell et al, 2014 ; Jeckelmann and Fotiadis, 2019 ; 2020 ; Kantipudi et al, 2020 ; Kantipudi et al, 2021 ; Kantipudi and Fotiadis, 2021 ; Jeckelmann et al, 2022 ).…”

Section: Introductionmentioning

confidence: 99%

Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay

Kantipudi

Harder²,

Fotiadis³

2023

Front. Physiol.

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Amino acids have diverse and essential roles in many cellular functions such as in protein synthesis, metabolism and as precursors of different hormones. Translocation of amino acids and derivatives thereof across biological membranes is mediated by amino acid transporters. 4F2hc-LAT1 is a heterodimeric amino acid transporter that is composed of two subunits belonging to the SLC3 (4F2hc) and SLC7 (LAT1) solute carrier families. The ancillary protein 4F2hc is responsible for the correct trafficking and regulation of the transporter LAT1. Preclinical studies have identified 4F2hc-LAT1 as a valid anticancer target due to its importance in tumor progression. The scintillation proximity assay (SPA) is a valuable radioligand binding assay that allows the identification and characterization of ligands of membrane proteins. Here, we present a SPA ligand binding study using purified recombinant human 4F2hc-LAT1 protein and the radioligand [3H]L-leucine as tracer. Binding affinities of different 4F2hc-LAT1 substrates and inhibitors determined by SPA are comparable with previously reported Km and IC50 values from 4F2hc-LAT1 cell-based uptake assays. In summary, the SPA is a valuable method for the identification and characterization of ligands of membrane transporters including inhibitors. In contrast to cell-based assays, where the potential interference with other proteins such as endogenous transporters persists, the SPA uses purified protein making target engagement and characterization of ligands highly reliable.

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Heterologous (Over) Expression of Human SoLute Carrier (SLC) in Yeast: A Well-Recognized Tool for Human Transporter Function/Structure Studies

Cited by 3 publications

References 241 publications

Deciphering the Diversity in Bacterial Transporters That Salvage Queuosine Precursors

Deciphering the Diversity in Bacterial Transporters That Salvage Queuosine Precursors

Deciphering the Diversity of Bacterial Transporters that Salvage Queuosine Precursors

Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay

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