1. Neuroscience

Localization, proteomics, and metabolite profiling reveal a putative vesicular transporter for UDP-glucose

  1. Cheng Qian
  2. Zhaofa Wu
  3. Rongbo Sun
  4. Huasheng Yu
  5. Jianzhi Zeng
  6. Yi Rao
  7. Yulong Li  Is a corresponding author
  1. School of Life Sciences, Tsinghua University, China
  2. State Key Laboratory of Membrane Biology, Peking University School of Life Sciences, China
  3. PKU-IDG/McGovern Institute for Brain Research, China
  4. Peking-Tsinghua Center for Life Sciences, China
  5. Chinese Institute for Brain Research, China
https://doi.org/10.7554/eLife.65417
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Cite this article
  1. Cheng Qian
  2. Zhaofa Wu
  3. Rongbo Sun
  4. Huasheng Yu
  5. Jianzhi Zeng
  6. Yi Rao
  7. Yulong Li
(2021)
Localization, proteomics, and metabolite profiling reveal a putative vesicular transporter for UDP-glucose
eLife 10:e65417.
https://doi.org/10.7554/eLife.65417
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7 figures, 1 table and 4 additional files

Figures

Figure 1 with 1 supplement
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Localization profiling of SLC family members reveals candidate vesicular transporters.

(A) Top: Schematic diagram of the localization profiling strategy. Red and green fluorescent signals were collected using confocal microscopy imaging of cultured rat neurons co-expressing …

Figure 1—source data 1

Genes for co-localization quantification.

https://cdn.elifesciences.org/articles/65417/elife-65417-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
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Subcellular localization of SLC35D3 at a reduced expression level.

(A) Schematic diagram of the epitope-tagged SLC35D3. (B) Left: Representative images of cultured neurons overexpressing SLC35D3-EGFP (green) by: plasmid transfection, lentivirus infection, and …

Figure 2 with 2 supplements
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Proteomics profiling of SVs identifies novel putative vesicular SLC transporters.

(A) Schematic diagram depicting the strategy for proteomics profiling of SVs immunoisolated from fractionated mouse brain homogenates. (B) Top: western blot analysis of the indicated protein markers …

Figure 2—source data 1

Proteomics profiling of SVs.

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Repeatability of the proteomic data Scatterplots showing the correlation between independent biological trials.
Figure 2—figure supplement 2
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SLC35D3 in subcellular fractionation of SVs.

(A) Schematic diagram depicting the strategy for subcellular fractionation of SVs from mouse brain homogenates. (B) Top: western blot analysis of SLC35D3-EGFP-3xFlag and the indicated protein …

Figure 3
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Validation of the vesicular localization of SLC35D3 using electron microscopy.

(A) Schematic diagram depicting the APEX2-based labeling strategy for studying ultrastructural localization. (B–E) Representative EM images (left) and distribution of organelle darkness (right) of …

Figure 3—source data 1

Quantification of organelle darkness.

https://cdn.elifesciences.org/articles/65417/elife-65417-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
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The targeted metabolite profiling reveals putative substrates of SLC35D3.

(A) Phylogenic tree of the SLC35 transporter family and known corresponding substrates. SLC35A2 and SLC35D3 are shown in blue and green, respectively. O: orphan transporters. (B) Left: …

Figure 4—source data 1

Targeted metabolite profiling of nucleotide sugars.

https://cdn.elifesciences.org/articles/65417/elife-65417-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
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Additional analysis of metabolite profiling.

(A) Representative brightfield (BF) and fluorescence (GFP) images of control (SLC35A2KO) cells and cells overexpressing EGFP-tagged SLC35A2 (SLC35A2-EGFP) or SLC35D3 (SLC35D3-EGFP). Scale bar: 10 …

Figure 5 with 1 supplement
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Validation and characterization of the UDP-glucose transport activity of SLC35D3.

(A) Schematic diagram depicting the transport assay using organelles isolated from HEK293T cells. (B) Summary of the transport of [3H]-UDP-glc, [3H]-UDP-gal, and [3H]-UDP-glcNAc (500 nM each) in …

Figure 5—source data 1

UDP-glucose transport activity of SLC35D3.

https://cdn.elifesciences.org/articles/65417/elife-65417-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
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Pharmacology-insensitive transport activity by SLC35A3.

(A) Summary of the transport of [3H]-UDP-glc and [3H]-UDP-glcNAc (500 nM each) in control (HEK293T) cells and in cells overexpressing human SLC35A3 (hSLC35A3), n = 3 experiments each. p=2.62E-5 in …

Figure 6
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Working model depicting SLC35D3 as a putative UDP-glucose transporter on SVs.

SLC35D3 is a vesicular transporter which potentially mediate transport of UDP-glucose into SVs. UDP-glucose may function as a signaling molecule through a GPCR namely P2Y14.

Author response image 1
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Test of anti-SLC35D3 antibodies (A) Representative images of cultured neurons expressing SYP-EGFP (green) or SLC35D3-mCherry (red) immunostained with anti-EGFP or anti-SLC35D3 antibodies (magenta), respectively.

Arrowheads indicate SYP-EGFP positive puncta and arrows indicate SLC35D3 positive puncta. Scale bars: 10 μm. (B) Left: Representative images of HEK293T cells with/without transfection of …

Tables

Key resources table
Reagent type
(species) or resource		DesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)SYPGenBankNM_009305.2
Gene (Homo sapiens)SLC35A2GenBankNM_005660.3
Gene (Mus musculus)SLC35D3GenBankBC139194.1
Gene (Homo sapiens)SLC35D3GenBankKJ896073.1
Strain, strain background (Mus musculus)Wild-typeCharles RiverC57BL6/J, RRID:MGI:5650797
Strain, strain background (AAV)AAV-PhP.eB hSyn-SLC35D3-EGFP-3xFlagVigeneTiter: 7.68 × 1013 gc/ml
Cell line (Homo sapiens)HEK293TATCCRRID:CVCL_0063
Cell line (Homo sapiens)SLC35A2KO cell lineThis paperMaterials and methods section, ‘KO cell line establishment and validation’
AntibodyPolyclonal rabbit anti-VGLUT1Synaptic SystemsCat. #: 135302 RRID:AB_887877WB dilution 1:1000
AntibodyPolyclonal rabbit anti-VGLUT2Synaptic SystemsCat. #: 135402 RRID:AB_2187539WB dilution 1:1000
AntibodyMonoclonal mouse anti-SYPSynaptic SystemsCat. #: 101011 RRID:AB_887824WB and IF dilution 1:1000
AntibodyPolyclonal rabbit anti-SYPCell Signaling TechnologyCat. #: 5461 RRID:AB_10698743WB dilution 1:1000
AntibodyMonoclonal mouse anti-VAMP2Synaptic SystemsCat. #: 104211 RRID:AB_887811WB dilution 1:1000
AntibodyMonoclonal mouse anti-PSD95NeuroMabCat. #: 75–028 RRID:AB_2292909WB dilution 1:1000
AntibodyMonoclonal mouse anti-FlagSigma-AldrichCat. #: F9291 RRID:AB_439698WB dilution 1:1000
AntibodyPolyclonal chicken anti-GFPAbcamCat. #: Ab13970 RRID:AB_300798IF dilution 1:1000
AntibodyMonoclonal rabbit anti-CALRCell Signaling TechnologyCat. #: 12238 RRID:AB_2688013WB dilution 1:1000
AntibodyPolyclonal rabbit anti-GM130Cell Signaling TechnologyCat. #: 12480 RRID:AB_2797933WB dilution 1:500
AntibodyPolyclonal rabbit anti-Chg ASynaptic SystemsCat. #: 259003 RRID:AB_2619972WB and IF dilution 1:500
Recombinant DNA reagentpN3-human SLC35D3-mCherry (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpN3-mouse SYP-EGFP (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpN3-rat VGLUT1-APEX2 (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpN3-OMM-APEX2 (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpN3-human SLC35D3-APEX2 (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpPacific-mouse SLC35D3-EGFP (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpPacific-human SLC35D3-EGFP (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpPacific-human SLC35A2-EGFP (Plasmid)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpAAV-hSyn-human SLC35D3-EGFP-3xFlag (AAV vector)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentpLenti hSyn-human SLC35D3-EGFP-3xFlag (lenti vector)This paperMaterials and methods section, ‘Molecular biology’
Recombinant DNA reagentHuman ORFeome 8.1Center for Cancer Systems Biologyhttp://horfdb.dfci.harvard.edu/Full-length human cDNAs
Recombinant DNA reagentDNASUNIGMS-funded Protein Structure Initiative (PSI)https://dnasu.org/DNASU/Home.doFull-length human cDNAs
Recombinant DNA reagentThe PlasmID RepositoryDF/HCC DNA Resource Core at Harvard Medical Schoolhttps://plasmid.med.harvard.edu/PLASMID/Home.xhtmlFull-length human cDNAs
Chemical compound, drugUDP-glucoseSanta CruzCat. #: sc-296687
Chemical compound, drugUDP-galactoseSanta CruzCat. #: sc-286849A
Chemical compound, drugUDP-N-acetylgalactosamineSigma-AldrichCat. #: U5252
Chemical compound, drugUDP-N-acetylglucosamineSigma-AldrichCat. #: U4375
Chemical compound, drugUDP-xyloseSugarsTechCat. #: SN02004
Chemical compound, drugUDP-glucuronic acidSanta CruzCat. #: sc-216043
Chemical compound, drugCMP-sialic acidSigma-AldrichCat. #: C8271
Chemical compound, drugGDP-fucoseSanta CruzCat. #: sc-221696A
Chemical compound, drugGDP-mannoseSanta CruzCat. #: sc-285856A
Chemical compound, drugUridine diphosphate glucose [6–3H]PerkinElmerCat. #: NET1163250UC
Chemical compound, drugUridine diphosphate galactose [1–3H]ARCCat. #: ART0737
Chemical compound, drugUridine diphosphate N-acetylglucosamine [6–3H]ARCCat. #: ART0128
Chemical compound, drugValinomycinSigma-AldrichCat. #: V0627
Chemical compound, drugNigericinSigma-AldrichCat. #: N7143
Chemical compound, drugFCCPSigma-AldrichCat. #: C2920
Chemical compound, drugN-EthylmaleimideSigma-AldrichCat. #: E3876
Chemical compound, drugBafilomycin A1abcamCat. #: ab120497
OtherProtein G dynabeadsThermoCat. #: 10004D

Additional files

Source code 1

in silica Puncta Analyzer tool.

https://cdn.elifesciences.org/articles/65417/elife-65417-code1-v2.zip
Supplementary file 1

Vesicular transporters identified in SLC localization profiling.

https://cdn.elifesciences.org/articles/65417/elife-65417-supp1-v2.docx
Supplementary file 2

SLC transporters enriched in immunoisolated synaptic vesicles.

https://cdn.elifesciences.org/articles/65417/elife-65417-supp2-v2.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/65417/elife-65417-transrepform-v2.docx

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