参见作者原研究论文

本实验方案简略版
Feb 2020

本文章节


 

Polyamine Transport Assay Using Reconstituted Yeast Membranes
利用重构酵母细胞膜的多胺转运实验   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

ATP13A2/PARK9 is a late endo-/lysosomal P5B transport ATPase that is associated with several neurodegenerative disorders. We recently characterized ATP13A2 as a lysosomal polyamine exporter, which sheds light on the molecular identity of the unknown mammalian polyamine transport system. Here, we describe step by step a protocol to measure radiolabeled polyamine transport in reconstituted vesicles from yeast cells overexpressing human ATP13A2. This protocol was developed as part of our recent publication (van Veen et al., 2020) and will be useful for characterizing the transport function of other putative polyamine transporters, such as isoforms of the P5B transport ATPases.

Keywords: Polyamine (多胺), Spermine (精胺), Transport assay (运输试验), Reconstitution (重构), Yeast membranes (酵母细胞膜), P5 ATPase (P5 ATPase), ATP13A2 (ATP13A2)

Background

ATP13A2/PARK9 encodes a ubiquitously expressed late endo-/lysosomal membrane protein that is implicated in a spectrum of neurodegenerative disorders, like early-onset Parkinson’s disease (Di Fonzo et al., 2007; Lin et al., 2008) and Kufor-Rakeb syndrome (an early-onset parkinsonism with dementia) (Ramirez et al., 2006; Park et al., 2011). ATP13A2 belongs to the P-type transport ATPases, a family of active transporters that transiently form a phospho-intermediate as a consequence of ATP hydrolysis (Kuhlbrandt, 2004). ATP13A2 is a member of the P5 subfamily, which was identified by genome sequencing more than twenty years ago (Axelsen and Palmgren, 1998) and contains five human isoforms (ATP13A1-5). However, the transported substrate of ATP13A2 remained unknown until recently, when we characterized ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine (SPM) (van Veen et al., 2020). ATP13A2 is activated by two regulatory lipids, phosphatidic acid and phosphatidylinositol(3,5)bisphosphate (Holemans et al., 2015; van Veen et al., 2020). To prove that ATP13A2 transports polyamines over the membrane, we developed a transport assay using tritium-labelled SPM ([3H]-SPM) and yeast membrane-derived vesicles recombinantly expressing human ATP13A2 with C-terminal BAD (biotin acceptor domain) tag (ATP13A2-BAD).


The polyamine transport assay starts with the production of reconstituted vesicles from solubilized yeast membranes that contain overexpressed ATP13A2-BAD, in the presence of the lipids phosphatidylcholine and phosphatidic acid. The generated proteoliposomes will contain both right-side-out and inside-out oriented reconstituted ATP13A2 (Figure 1), and we made use of this principle to set up a transport assay where we follow the luminal accumulation of [3H]-SPM in the vesicles. Since ATP13A2 is a lysosomal exporter, ATP13A2 should be inserted inside-out (cytosolic domains facing the lumen) to allow luminal accumulation of [3H]-SPM. In this orientation, the lumen of the proteoliposomes should be supplemented with ATP to ensure that ATP binding and ATPase activity can occur at the nucleotide-binding domain. Therefore, we reconstitute the proteoliposomes in the presence of ATP and an ATP regenerating system (phosphocreatine/creatine phosphokinase). In the inside-out orientation, ATP13A2 will promote the uptake of [3H]-SPM in the proteoliposomes if ATP is present inside, in line with ATP13A2-mediated polyamine transport from the extra-cytosol to the cytosol in a cellular context (Figure 1).



Figure 1. Graphical representation of the principle behind the polyamine transport assay. To assay polyamine transport, we use proteoliposome vesicles, reconstituted from yeast membranes that contain ATP13A2. The generated proteoliposomes will contain both right-side-out and inside-out oriented reconstituted ATP13A2. To allow luminal accumulation of [3H]-SPM, ATP13A2 should be inserted inside-out (cytosolic domains facing the lumen) as ATP13A2 is a lysosomal exporter. In addition, the lumen of the proteoliposomes should be supplemented with ATP as in P-type ATPases, ATP binding occurs at a nucleotide-binding site located in one of the cytosolic domains. Only when ATP is present inside the proteoliposomes, [3H]-SPM accumulates within the vesicles, in line with ATP13A2’s cellular role as a lysosomal polyamine exporter.


Our protocol using yeast-derived membranes offers several advantages for the polyamine transport analysis as compared to mammalian systems (e.g., Uemura and Gerner, 2011). Although the preparation of yeast crude extract is more laborious and time-consuming compared to mammalian cell lysis, the benefit lies in the fact that yeast cells grow fast and are easy to culture with low cost and high yield of biomass. Moreover, yeast is a malleable model organism that is easily genetically manipulated. Furthermore, our protocol represents a clean in vitro technique as opposed to a cellular polyamine uptake assay. Our polyamine transport assay is applicable for other candidate polyamine transporters, which will help to establish the molecular players of the mammalian polyamine transport system, which remain largely unknown. Based on the high conservation of the substrate binding domain in the transmembrane helix M4, it is very likely that other mammalian P5B ATPases (ATP13A3-5) also play a role in the polyamine transport system, possibly with a slightly different substrate specificity, subcellular localization and/or tissue distribution. Therefore, our transport assay will also be valuable for characterizing the transport function of the other related P5B ATPases.


Materials and Reagents

Notes:

  1. All materials and reagents are kept at room temperature unless otherwise described. For the shelf life and storage temperature of reagents, we refer directly to the manufacturer’s instructions.

  2. Equivalent materials and reagents of other companies might also be suitable.


  1. Pipette tips

    P10 tips (Sarstedt, catalog number: 70.1130.20)

    P200 tips (Sarstedt, catalog number: 70.760.102)

    P1000 tips (VWR, catalog number: 613-0738)

  2. Filter pipette tips

    P10 filter tips (Greiner, catalog number: 771288)

    P200 filter tips (Greiner, catalog number: 739288)

    P1000 filter tips (Greiner, catalog number: 740288)

  3. Eppendorf tubes (Greiner, catalog number: 616201)

  4. Falcon tubes (Greiner, catalog numbers: 188271 [15 ml], 227261 [50 ml])

  5. Nitrile gloves (VWR, catalog number: 112-2371)

  6. Duran glass bottle (VWR, catalog numbers: 215-1516 [500 ml], 215-1517 [1,000 ml])

  7. Culture flasks (DWK Life Sciences, catalog number: 217715407)

  8. Syringe-driven filter unit (Merck, Millex, catalog number: SLGS033SB)

  9. Petri dishes (60 mm) (ThermoFisher, catalog number: 123-17)

  10. Bottle top vacuum filtration system (VWR, complete filtration unit with 0.2 µm pore size, catalog number: 514-0334)

  11. Acid-washed glass beads (Sigma-Aldrich, catalog number: G8772)

  12. Test tubes Soda glass (VWR, catalog number: 212-0013)

  13. Membrane filters, 0.45 μm pore size (Millipore, catalog number: HAWP02500)

  14. Saccharomyces cerevisiae strain W303-1B/Gal4-∆Pep4 (leu2-3, his3-11,15, trp1-1::TRP1-GAL10-GAL4, ura3-1, ade2-1, canr, cir+, ∆Pep4 MATα)

    Note: Strain available from corresponding author upon request.

  15. Plasmid DNA

    We use a pYeDP60 vector (with 2-μm circle replication origin, URA3 and ADE2 selection markers, and a galactose inducible promoter) containing a yeast codon-optimized version of human ATP13A2 variant 2 cDNA followed by a thrombin cleavage site and a C-terminal BAD tag (Jidenko et al., 2006; Azouaoui et al., 2014) (Figure 2). In our experiments, we use a catalytically inactive ATP13A2 variant, namely the E343A mutant (van Veen et al., 2020), as a negative transport control. E343 is positioned in the catalytic site for dephosphorylation, which is highly conserved among P-type ATPases (341TGES motif in human ATP13A2 isoform 2).

    Note: Plasmids available from corresponding author upon request.



    Figure 2. Schematic representation of the ATP13A2 expression plasmid map. The expression plasmid for ATP13A2 is a modified pYeDP60 plasmid. The pYeDP60 vector contains the 2-μm circle replication origin, the URA3 and ADE2 selection markers, a galactose inducible GAL10/CYC1 promoter, multiple cloning sites, and the PGK1 terminator. The human ATP13A2 gene was cloned into the plasmid with a thrombin cleavable BAD (biotin acceptor domain)-tag (Jidenko et al., 2006; Azouaoui et al., 2014).


  16. Yeast extract granulated (Merck, catalog number: 1.03753.0500)

  17. Peptone from casein (Tryptone) (Merck, catalog number: 1.07213.2500)

  18. D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270)

  19. Cuvettes (VWR, catalog number: 634-0676)

  20. Tris base (Sigma-Aldrich, Trizma base, catalog number: T1503)

  21. Ethylenediaminetetra-acetic acid (EDTA) (BDH Laboratory Supplies, catalog number: 280214S)

  22. Hydrochloric acid (HCl) fuming 37% (Merck, catalog number: 1.00317.2501)

  23. HCl 0.5 N (Reagecon, catalog number: H20501)

  24. Lithium acetate dihydrate (LiAc) (Sigma-Aldrich, catalog number: L6883)

  25. Acetic acid (Sigma-Aldrich, catalog number: 27225)

  26. DNA from herring sperm (Sigma-Aldrich, catalog number: D7290)

  27. Polyethylene glycol (PEG) (Sigma-Aldrich, catalog number: P4338)

  28. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855)

  29. Bacto Agar (BD, catalog number: 214010)

  30. Yeast dropout mix without uracil (Sigma-Aldrich, catalog number: Y1501)

  31. Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626)

  32. Ethanol absolute (VWR, catalog number: 20821.296)

  33. D-(+)-Galactose (Sigma-Aldrich, catalog number: G0625)

  34. KCl (Sigma-Aldrich, catalog number: P9541)

  35. Sorbitol (Sigma-Aldrich, catalog number: S1876)

  36. NaOH (Merck, catalog number: 6498.1000)

  37. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 93482)

  38. Protease inhibitor (Sigma-Aldrich, SIGMAFAST Protease Inhibitor Cocktail Tablets, catalog number: S8830)

  39. HEPES (Sigma-Aldrich, catalog number: H3375)

  40. Sucrose (Sigma-Aldrich, catalog number: S7903)

  41. CaCl2 (Sigma-Aldrich, catalog number: C3881)

  42. n-Dodecyl-β-D-Maltopyranoside (DDM) (Inalco, catalog number: 1758-1350)

  43. Bradford reagent (Sigma-Aldrich, catalog number: B6916)

  44. Egg phosphatidylcholine (Avanti Polar Lipids, L-α-phosphatidylcholine [Egg, Chicken], catalog number: 840051C)

  45. 18:1 phosphatidic acid (Avanti Polar Lipids, 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), catalog number: 840875C)

  46. Bio-Beads SM-2 Adsorbents (BioRad, catalog number: 1523920)

  47. ATP disodium salt (Roche Diagnostics, catalog number: 10127531001)

  48. MgCl2 hexahydrate (Sigma-Aldrich, catalog number: M2670)

  49. Phosphocreatine disodium salt hydrate (Sigma-Aldrich, catalog number: P7936)

  50. Creatine phosphokinase (Sigma-Aldrich, catalog number: C3755)

  51. Anti-ATP13A2 antibody (Sigma-Aldrich, catalog number: A3361)

  52. Anti-rabbit IgG, HRP-linked antibody (Bioké, catalog number: 7074S)

  53. γ-(N-Morpholino)propanesulphonic acid (MOPS) (VWR, catalog number: A1076.1000)

  54. KOH (Sigma-Aldrich, catalog number: 221473)

  55. Dithiothreitol (DTT) (VWR, catalog number: A2948.0025)

  56. SPM (Sigma-Aldrich, catalog number: 85590)

  57. [3H]-SPM, 1 mCi/ml, 20 µM (American Radiolabeled Chemicals, Inc.; catalog number: ART0471)

  58. Liquid scintillation cocktail (MP Biomedicals, Ecolite(+)TM Liquid Scintillation Fluid, catalog number: 882475)

  59. Chloroform stock (Sigma-Aldrich, catalog number: C2432)

  60. 20% glucose (see Recipe 1)

  61. YPD-agar plates (see Recipe 2)

  62. YPD medium (see Recipe 3)

  63. 10x TE stock (see Recipe 4)

  64. 10x LiAc stock (see Recipe 5)

  65. 1x TE/LiAc solution (see Recipe 6)

  66. Single-stranded herring sperm DNA (see Recipe 7)

  67. 50% PEG w/v (see Recipe 8)

  68. PEG solution (see Recipe 9)

  69. SD-uracil agar plates (see Recipe 10)

  70. SD-uracil medium (see Recipe 11)

  71. YPGE2x medium (see Recipe 12)

  72. 20% galactose (see Recipe 13)

  73. TEKS buffer (see Recipe 14)

  74. TESin buffer (see Recipe 15)

  75. HS buffer (see Recipe 16)

  76. Buffer T (see Recipe 17)

  77. Buffer T/DDM (see Recipe 18)

  78. Buffer T/lipid mix (see Recipe 19)

  79. 0.1 M ATP (see Recipe 20)

  80. 0.1 M MgCl2 (see Recipe 21)

  81. 0.2 M phosphocreatine (see Recipe 22)

  82. ATP-regenerating system (see Recipe 23)

  83. 500 mM DTT (see Recipe 24)

  84. 4x reaction buffer (see Recipe 25)

  85. 1x reaction buffer (see Recipe 26)

  86. 0.1 M MOPS (pH 7.0) (see Recipe 27)

  87. 10 mM SPM (see Recipe 28)

  88. 100 µl [3H]-SPM/unlabeled SPM mix (see Recipe 29)

Equipment

  1. Pipettes

    P2 pipette (Gilson, catalog number: FA10001M)

    P10 pipette (Gilson, catalog number: FA10002M)

    P100 pipette (Gilson, catalog number: FA10004M)

    P200 pipette (Gilson, catalog number: FA10005M)

    P1000 pipette (Gilson, catalog number: FA10006M)

  2. Serological pipettor (Sigma, BRAND® accu-jet® pro pipette controller, catalog number: Z637637)

  3. Serological pipettes

    5 ml (Sarstedt, catalog number: 86.1253.001)

    10 ml (Sarstedt, catalog number: 86.1254.001)

    25 ml (Sarstedt, catalog number: 86.1685.001)

  4. Ultracentrifuge tubes for Ti45 rotor (Beckman Coulter, catalog number: 355655)

  5. Ultracentrifuge tubes for Ti70 rotor (Beckman Coulter, catalog number: 355630)

  6. Glass Büchner filter funnel (Millipore, catalog number: XX1014700)

  7. Magnetic stirrer (Heidolph Instruments, MR Hei-Standard, catalog number: 505-20000-00)

  8. Refrigerated incubator with shaker (New Brunswick Scientific, model: Innova 4230)

  9. Ice bucket (e.g., styrofoam box)

  10. Autoclave (LTE Scientific Ltd, Series 100 Autoclave)

  11. MilliQ (MQ) water system (Sartorius, Arium Pro)

  12. Spectrophotometer (Beckman Coulter, model number: DU-640B)

  13. Centrifuge (Eppendorf, model: 5804 R) with rotor (Eppendorf, model: A-4-44)

  14. Microcentrifuge (Eppendorf, Centrifuge 5417 R) with rotor (Eppendorf, model: F45-30-11)

  15. Vortex (VWR, model: Vortex-Genie® 2, catalog number: 444-5900)

  16. Thermomixer (Eppendorf, model: Thermomixer Comfort)

  17. Water bath (Memmert)

  18. Homogenizer (BioSpec products, BeadBeater, catalog number: 1107900EUR)

  19. Pressure vacuum pump (Gelman Sciences, Gelman Little Giant, model: 13156)

  20. Hamilton syringes (1 ml) (VWR, 1001 LTN, catalog number: 613-1300)

  21. Nitrogen gas blow-down system (made in-house)

  22. Ultracentrifuge (Beckman Coulter, model: Optima XPN-90)

  23. Ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti)

  24. Ultracentrifuge rotor (Beckman Coulter, model: Type 90 Ti)

  25. Head-over-head rotator (Labinco BV, L28 Test-Tube Rotator, catalog number: 28000)

  26. Vacuum filtration manifold (Millipore, catalog number: XX2702550)

  27. Liquid scintillation analyzer (Perkin Elmer, TRI-CARB 2900TR)

Procedure

  1. Yeast transformation

    We transformed the yeast strain W303-1B/Gal4-∆Pep4 (leu2-3, his3-11,15, trp1-1::TRP1-GAL10-GAL4, ura3-1, ade2-1, canr, cir+, ∆Pep4 MATα) with the pYeDP60 vector containing hATP13A2 WT or the catalytically dead E343A mutant according to the lithium acetate/single-stranded carrier DNA/polyethylene glycol method with minor modifications (Gietz and Woods, 2002).

    1. Plate the yeast from the glycerol stock on a YPD-agar plate and incubate for 48 h at 30 °C.

    2. Inoculate 20 ml YPD medium from the fresh plate and incubate overnight at 30 °C, 230 rpm in a shaking incubator.

    3. Measure the OD of the overnight culture at 600 nm (OD600).

      Note: 1 OD600 equals approximately 107 cells/ml.

    4. Dilute the culture to 0.1 OD600 in 10 ml YPD and incubate for 4-6 h at 30 °C, 230 rpm in a shaking incubator until the OD600 of the culture lies between 0.4 and 0.8.

    5. Spin down the cell amount equivalent to 2 OD600 (= ± 2 x 107 cells) in swinging buckets (700 x g; 5 min).

    6. Wash the pellet in 1 ml sterile MQ and resuspend with a pipet tip. Transfer to an Eppendorf tube.

    7. Spin down the cells (2,500 x g; 2 min) using a microcentrifuge.

    8. Resuspend pellet in 1 ml 1x TE/LiAc.

    9. Spin down the cells (2,500 x g; 2 min) using a microcentrifuge.

    10. Resuspend pellet in 100 µl 1x TE/LiAc.

    11. Add 10 µl of single-stranded herring sperm DNA.

    12. Add 1 to 5 µg of plasmid DNA.

    13. Vortex.

    14. Add 600 µl of PEG solution.

    15. Vortex.

    16. Incubate for 45 min at 30 °C (volume = approximately 730 µl).

    17. Add DMSO to a final concentration of 10% (approximately 73 µl).

    18. Heat shock at 42 °C for 15 min.

    19. Cool on ice for 1-2 min.

    20. Spin down the cells (2,500 x g; 2 min) using a microcentrifuge.

    21. Resuspend pellet in 200 µl of YPD and incubate for 2 h at 30 °C.

    22. Spin down the cells (2,500 x g; 2 min) using a microcentrifuge.

    23. Resuspend the cell pellet in sterile MQ and plate onto an SD-uracil agar plate.

    24. Incubate the plate for 48 h at 30 °C to recover transformants.


  2. Yeast culture

    Here, we followed a similar strategy as described before (Jidenko et al., 2006; Azouaoui et al., 2014) with minor modifications.

    1. Inoculate 20 ml of SD-uracil medium from the fresh plate (Step A24) to further select for yeast cells that carry the plasmid with URA3 selection marker and incubate for 24 h at 28 °C and 200 rpm in a shaking incubator.

    2. Measure the OD600 of the culture.

    3. Inoculate 100 ml of SD-uracil medium to a final OD600 of 0.2 and incubate for 12 h at 28 °C and 200 rpm in a shaking incubator.

    4. Measure the OD600 of the culture.

    5. Inoculate 3.6 L of YPGE2X medium to a final OD600 of 0.05 and incubate for 36 h at 28 °C and 175 rpm in a shaking incubator.

      Note: Nine 1,000 ml culture flasks containing 400 ml of yeast culture.

    6. Induce ATP13A2-BAD expression with 2% galactose (50 ml of 20% galactose solution per 500 ml culture) and incubate for 12 h at 18 °C and 175 rpm in a shaking incubator.

      Note: The temperature downshift from 28 °C to 18 °C will reduce the rate of protein synthesis and therefore, facilitate proper protein folding, increasing the yield of properly folded and functional overexpressed protein. In addition, the lower temperature will decrease protein degradation.

    7. Repeat galactose induction to ensure high overexpression levels of ATP13A2-BAD and incubate for another 12 h at 18 °C and 175 rpm in a shaking incubator.

    8. Spin down the yeast cells (1,000 x g; 10 min; 4 °C).

    9. Weigh the pellet.

      Note: The yield is typically 20-30 g/L yeast culture. The cell pellet can be processed directly or stored at -20 °C for a maximum of 2 weeks.


  3. Yeast membrane preparation

    1. In case yeast cell pellet was frozen, thaw on ice.

    2. Resuspend cell pellet in TEKS buffer (volume (ml) = approximately 2 x the weight of the cell pellet (g) as determined in step B9) and incubate for 15 min at 4 °C while mixing with a magnetic stirrer.

    3. Spin down the cells (1,000 x g; 10 min; 4 °C).

    4. Resuspend the pellet in TESin buffer (volume (ml) = approximately 1 x the weight of the cell pellet (g) as determined in Step B9).

    5. Break the yeast cells using a BeadBeater.

      1. Fill the chamber with 200 ml yeast suspension (add TES in buffer if not enough) and add 200 ml cold glass beads. Chamber should be as full as possible.

      2. Fill ice water jacket with crushed ice and water.

      3. Bead-beat in cold room for 5 min. Pause for 3 min after every min to reduce heating of the sample and BeadBeater.

      4. Recover crude extract using Büchner funnel and vacuum pump.

    6. Test the pH of the lysate by using pH paper and, if necessary, adjust the pH of the crude extract to pH 7.5 with saturated NaOH solution.

    7. Centrifuge the crude extract at 2,000 x g for 20 min (4 °C).

    8. Centrifuge the resulting supernatant at 20,000 x g for 20 min using ultracentrifuge rotor Type 45 Ti (4 °C).

    9. Centrifuge the resulting supernatant at 200,000 x g for 1 h using ultracentrifuge rotor Type 45 Ti (4 °C).

    10. Resuspend the resulting pellet (that is, the light membrane fraction, P3) in HS buffer.

      Note: The volume for resuspension should be determined by eye and be the minimal amount required to homogeneously dissolve the pellet.

    11. Determine the protein concentration of the P3 fraction using a classical Bradford assay (with known BSA concentrations as a protein standard).

      Note: The measured protein concentration typically ranges between 35-50 mg/ml. For downstream reconstitution purposes, a minimum yield of 70 mg total protein is required per experiment.

    12. Aliquot the P3 membranes per 2 ml and freeze in liquid N2.

    13. Store at -80 °C.

      Note: The membranes can be stored at -80 °C for a maximum of 6 months.


  4. Reconstitution of yeast membranes

    To reconstitute yeast membranes, we followed a similar strategy as described before (Papadopulos et al., 2007) with some modifications.

    1. Dilute 2 ml of the P3 membranes (thawed on ice) to 10 µg/µl in buffer T/DDM.

      Note: The amount of total protein should be at least 70 mg.

    2. Incubate for 45 min at 4 °C in a head-over-head rotator.

    3. Centrifuge for 30 min at 200,000 x g to pellet the insoluble fraction using ultracentrifuge rotor Type 90 Ti (4 °C).

    4. Supplement 2 ml of the supernatant, i.e., detergent extract, with an equal volume of buffer T/lipid mix and for the “ATP inside” condition, also an equal volume of ATP-regenerating system (Table 1):


      Table 1.Sample preparation for reconstitution


    5. Incubate the samples with 100 mg/ml Bio-Beads for 3 h at room temperature in a head-over-head rotator to remove the DDM and reconstitute proteoliposomes.

    6. Add 200 mg/ml Bio-Beads and incubate overnight at 4 °C in a head-over-head rotator.

    7. Centrifuge for 1 h at 200,000 x g using ultracentrifuge rotor Type 90 Ti (4 °C).

    8. Recover vesicles by resuspending the pellet in 2 ml of buffer T.

      Note: The measured protein concentration typically ranges between 1-5 mg/ml.

    9. Determine the protein concentration using a classical Bradford assay.

    10. Check reconstituted protein levels via Western blot (Figure 3).



      Figure 3. Immunoblot of yeast membranes and reconstituted vesicles. P3 membranes containing ATP13A2-BAD are solubilized by the detergent DDM. The detergent extract is supplemented with the lipids phosphatidylcholine and phosphatidic acid, and then treated with Bio-Beads to remove the DDM and reconstitute proteoliposomes (‘no ATP’). To generate proteoliposomes that contained intraluminal ATP (‘ATP inside’), we added ATP and an ATP-regenerating system before incubation with the Bio-Beads. Reconstituted ATP13A2-BAD protein levels are checked via Western blot analysis using primary anti-ATP13A2 antibody (1/1,000) and HRP-conjugated secondary antibodies (1/2,000). 12 µl of sample was loaded per lane.


  5. Polyamine transport assay

    Note:

    1. The polyamine transport assay should be performed in a designated radioactive area by authorized personnel and radioactive material handling precautions have to be undertaken. Gloves, lab goggles, lab coats and a dosimeter to monitor personal exposure should be worn at all times. The work should be performed behind Plexiglass screens and only pipette tips with filters should be used. The regulations of the institution should be followed for the storage of radioactive material, and disposal of solid and liquid radioactive waste.
    2. Measure [3H]-SPM uptake into freshly prepared vesicles within 60 min.


    1. Dilute the freshly prepared vesicles (“no ATP” or “ATP inside”) to 1 µg/µl in buffer T.

      Note: We typically use 1 ml of vesicles. At least 300 µg (“ATP inside” vesicles) – 600 µg (“no ATP” vesicles) is required to proceed to the next steps.

    2. Place four 0.45 µm Millipore filters on filtration manifold (Figure 4).

      Note: Filters are pre-wet in MQ. Use tweezers to hold them.

    3. Wash each filter with 4 ml 1x reaction buffer.

    4. Pipet the following into the reaction tubes for the different conditions (in duplo) (Table 2):


      Table 2. Sample preparation for transport assay


    5. Slightly vortex to thoroughly mix (avoid air bubble formation).

    6. Place the reaction tube in a 37 °C water bath for 5 min.

    7. Start the reaction by adding 100 µl [3H]-SPM/unlabeled SPM mix (final total SPM concentration: 1 mM).

    8. Take out 300 μl (45 µg vesicles) at chosen time points (0 min, 10 min) and pipet each aliquot on a different filter allowing to remove the liquid phase.

    9. Wash each filter with 4 ml 1x reaction buffer.

    10. Place the filters in the scintillation vials containing 7 ml scintillation liquid.

    11. Repeat steps for the remaining reaction tubes.

    12. Measure samples with a liquid scintillation counter.



      Figure 4. Experimental setup for polyamine transport assay. Filtration manifold (1) connected to a vacuum pump (2) with a collector for radioactive waste (3). Water bath (4).

Data analysis

The polyamine transport assay described measures the accumulation of [3H]-SPM, following a 10 min incubation period, in reconstituted vesicles that contain inside-out ATP13A2 and intraluminal ATP (“ATP inside” condition). The experiment is performed in parallel with different control conditions. Proteoliposomes with overexpressed ATP13A2 that only contain extraluminal ATP (“ATP outside” condition) or have no ATP at all (“no ATP” condition) serve as negative controls as these vesicles do not take up [3H]-SPM due to lack of ATP binding at the luminal nucleotide-binding domain of reconstituted inside-out ATP13A2 protein (Figure 1). In addition, reconstituted vesicles with overexpression of a catalytically inactive ATP13A2 variant (E343A) are included as a negative transport control. [3H]-SPM uptake is quantified by scintillation counting and values are normalized to 0 min for every condition. For each uptake time, duplicate determinations are made. A representative graph is shown in Figure 5.



Figure 5. Polyamine transport assay. Uptake of [3H]-SPM in yeast membrane-derived vesicles with overexpression of ATP13A2 WT vs. the catalytically dead mutant E343A (negative control). The condition ‘no ATP’ stands for reconstituted vesicles without ATP, whereas the conditions ‘ATP outside’ and ‘ATP inside’ represent proteoliposomes that contain extra- or intraluminal ATP and ATP-regenerating system, respectively (van Veen et al., 2020).

Notes

The here-described protocol can be expanded to perform the following types of measurements:

  1. Time dependency should be linear, which can be verified at various time points in the range of 0-30 min. The uptake rate can be calculated from the slope.

  2. A dose/response curve can be generated by testing a range of total SPM concentrations (the combination of cold and [3H]-SPM labeled together), in the physiological range of 0.01 µM-10 mM. The concentration dependency allows the determination of the apparent affinity for SPM (Km) and maximal turnover rate (Vmax) of the transporter. The kinetic parameters are calculated from the Hill equation with non-linear regression analysis, as in Holemans et al. (2014).

Recipes

  1. 20% glucose

    1. Put 500 ml MQ in a large beaker glass with a stir bar

    2. Add 200 g glucose

    3. Stir the solution for a few minutes

    4. Finalize volume to 1,000 ml with MQ

    5. Filter sterilize the solution

  2. YPD-agar plates

    Note: Plates should be stored at 4 °C in the dark.

    1. Put 3 g yeast extract, 6 g peptone and 6 g agar in a Duran glass bottle

    2. Add MQ up to 270 ml

    3. Autoclave at 121 °C for at least 30 min

    4. Add 30 ml 20% glucose

    5. Pour into Petri dishes

  3. YPD medium

    1. Put 10 g yeast extract and 20 g peptone in a Duran glass bottle

    2. Add MQ up to 900 ml

    3. Autoclave at 121 °C for at least 30 min

    4. Add 100 ml 20% glucose

  4. 10x TE stock (0.1 M Tris-HCl, pH 7.5, 0.01 M EDTA)

    0.606 g Tris

    0.146 g EDTA

    Add MQ up to 50 ml

    Adjust pH to 7.5 with HCl

    Filter sterilize the solution

  5. 10x LiAc stock (1 M LiAc, pH 7.5)

    5.101 g LiAc

    Add MQ up to 50 ml

    Adjust pH to 7.5 with diluted acetic acid

    Filter sterilize the solution

  6. 1x TE/LiAc solution

    Mix together 120 µl 10x TE stock, 120 µl 10x LiAc stock and 900 µl sterile MQ

  7. Single-stranded herring sperm DNA

    DNA is boiled for 20 min in water bath and then immediately cooled on ice

  8. 50% PEG w/v

    1. Weigh off 25 g PEG

    2. Add sterile MQ up to 50 ml

    3. Filter sterilize the solution

  9. PEG solution (40% PEG, 1x TE, 1x LiAc)

    Mix together 800 µl 50% PEG, 100 µl 10x TE stock and 100 µl 10x LiAc stock

  10. SD-uracil agar plates

    Note: Plates should be stored at 4 °C in the dark.

    1. Put 5 g agar, 0.475 g yeast dropout mix without uracil and 1.675 g yeast nitrogen base without amino acids in a Duran glass bottle

    2. Add MQ up to 200 ml

    3. Autoclave at 121 °C for at least 30 min

    4. Add 50 ml 20% glucose

    5. Pour into Petri dishes

  11. SD-uracil medium

    Note: Medium should be stored in the dark.

    1. Put 0.95 g yeast dropout mix without uracil and 3.35 g yeast nitrogen base without amino acids in a Duran glass bottle

    2. Add MQ up to 450 ml

    3. Autoclave at 121 °C for at least 30 min

    4. Add 50 ml 20% glucose

  12. YPGE 2x medium

    1. Put 20 g yeast extract and 20 g peptone in a Duran glass bottle

    2. Add MQ up to 725 ml

    3. Autoclave at 121 °C for at least 30 min

    4. Add 50 ml 20% glucose

    5. Add 27 ml ethanol

  13. 20% galactose

    1. Put 500 ml MQ in a large beaker glass with a stir bar

    2. Add 200 g galactose

    3. Heat up while mixing to get it into solution

    4. Finalize volume to 1,000 ml with MQ

    5. Filter sterilize the solution

  14. TEKS buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.1 M KCl, 0.6 M sorbitol)

    Note: Buffer should be stored at 4 °C.

    6.057 g Tris

    0.292 g EDTA

    7.456 g KCl

    109.302 g sorbitol

    Add MQ up to 1000 ml

    Adjust pH to 7.5 with HCl

  15. TESin buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.6 M sorbitol, 1 mM PMSF, protease inhibitor cocktail)

    Note: Buffer should be stored at 4 °C.

    6.057 g Tris

    0.292 g EDTA

    109.302 g sorbitol

    Add MQ up to 1,000 ml

    Adjust pH to 7.5 with HCl

    Add Sigma Fast protease inhibitor cocktail on the day of the experiment. For 50 ml of buffer, add 1 tablet

    PMSF is added just before use. For 50 ml of buffer, add 500 µl of 100 mM PMSF

  16. HS buffer (20 mM HEPES-Tris, pH 7.4, 0.3 M sucrose, 0.1 mM CaCl2)

    Note: Buffer should be stored at 4 °C.

    0.477 g HEPES

    10.269 g sucrose

    0.0015 g CaCl2

    Add MQ up to 100 ml

    Adjust pH to 7.4 with Tris

  17. Buffer T (10 mM Tris-HCl, pH 7.4 and 1 mM EDTA)

    Note: Buffer should be stored at 4 °C.

    0.121 g Tris

    0.029 g EDTA

    Add MQ up to 100 ml

    Adjust pH to 7.4 with HCl

  18. Buffer T/DDM (buffer T supplemented with 1.4% DDM w/v)

    20 ml buffer T

    280 mg DDM

  19. Buffer T/lipid mix (buffer T containing 0.7% DDM w/v, 4.5 mM egg phosphatidylcholine and 0.5 mM 18:1 phosphatidic acid (PA)

    1. Add 173 mg egg phosphatidylcholine (6.92 ml of 25 mg/ml chloroform stock) to a glass tube and dry under nitrogen stream to make a lipid film

    2. Add 18 mg 18:1 PA (1.8 ml of 10 mg/ml chloroform stock) to the lipid film and dry under nitrogen stream

    3. Resolubilize the lipid film in 50 ml buffer T supplemented with 0.35 g DDM

  20. 0.1 M ATP

    0.551 g ATP

    Add MQ up to 10 ml

    Aliquot and store at -20 °C

  21. 0.1 M MgCl2

    0.203 g MgCl2

    Add MQ up to 10 ml

  22. 0.2 M phosphocreatine

    1 g phosphocreatine

    Add MQ up to 19.6 ml

    Aliquot and store at -20 °C

  23. ATP regenerating system

    200 µl 0.1 M ATP

    200 µl 0.1 M MgCl2

    200 µl 0.2 M phosphocreatine

    200 µl creatine phosphokinase (1 U/µl)

    1.2 ml MQ

  24. 500 mM DTT

    0.771 g DTT

    Add MQ up to 10 ml

    Aliquot and store at -20 °C

  25. 4x reaction buffer (200 mM MOPS, 400 mM KCl, 44 mM MgCl2, 4 mM DTT)

    Note: Buffer should be stored at 4 °C.

    4.184 g MOPS

    2.982 g KCl

    0.894 g MgCl2

    Add MQ up to 100 ml

    Adjust pH to 7.0 with KOH

    Add DTT just before use. For 5 ml of buffer, add 40 µl of 500 mM stock solution

  26. 1x reaction buffer (50 mM MOPS, 100 mM KCl, 11 mM MgCl2, 1 mM DTT)

    100 ml 4x reaction buffer

    300 ml MQ

  27. 0.1 M MOPS (pH 7.0)

    2.093 g MOPS

    Add MQ up to 100 ml

    Adjust pH to 7.0 with KOH

  28. 10 mM SPM

    0.020 g SPM

    Add 0.1 M MOPS (pH 7.0) up to 10 ml

    Aliquot and store under nitrogen gas at -80 °C. Once thawed, aliquots are not reused

  29. [3H]-SPM/unlabeled SPM mix (10 µCi, 0.2 µM [3H]-SPM, 10 mM SPM, total SPM concentration: 9.9 mM)

    10 µl [3H]-SPM

    990 µl 10 mM SPM

Acknowledgments

The described protocol was originally published as a method in van Veen et al., 2020. This work was funded by the Fonds Wetenschappelijk Onderzoek (FWO, Research Foundation Flanders) (G094219N, SBO Neuro-TRAFFIC S006617N and 1503117N), the KU Leuven (LysoCaN C16/15/073) and the Queen Elisabeth Medical Foundation for Neurosciences, Valine de Spoelberch Award. S.v.V. is an aspirant FWO research fellow (11Y7518N). We thank Dr. J. Lyons, University of Aarhus, for his help with the generation of the ATP13A2 BAD-tag fusion construct.

Competing interests

The authors declare no financial or non-financial competing interests.

References

  1. Axelsen, K. B. and Palmgren, M. G. (1998). Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46(1): 84-101.
  2. Azouaoui, H., Montigny, C., Ash, M. R., Fijalkowski, F., Jacquot, A., Gronberg, C., Lopez-Marques, R. L., Palmgren, M. G., Garrigos, M., M., le Maire, Decottignies, P., Gourdon, P., Nissen, P., Champeil, P. and Lenoir, G. (2014). A high-yield co-expression system for the purification of an intact Drs2p-Cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate. PLoS One 9(11): e112176.
  3. Di Fonzo, A., Chien, H. F., Socal, M., Giraudo, S., Tassorelli, C., Iliceto, G., Fabbrini, G., Marconi, R., Fincati, E., Abbruzzese, G., Marini, P., Squitieri, F., Horstink, M. W., Montagna, P., Libera, A. D., Stocchi, F., Goldwurm, S., Ferreira, J. J., Meco, G., Martignoni, E., Lopiano, L., Jardim, L. B., Oostra, B. A., Barbosa, E. R., N., Italian Parkinson Genetics and Bonifati, V. (2007). ATP13A2 missense mutations in juvenile parkinsonism and young onset Parkinson disease. Neurology 68(19): 1557-1562.
  4. Gietz, R. D. and Woods, R. A. (2002). Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350: 87-96.
  5. Holemans, T., Sorensen, D. M., van Veen, S., Martin, S., Hermans, D., Kemmer, G. C., Van den Haute, C., Baekelandt, V., Gunther Pomorski, T., Agostinis, P., Wuytack, F., Palmgren, M., Eggermont, J. and Vangheluwe, P. (2015). A lipid switch unlocks Parkinson's disease-associated ATP13A. Proc Natl Acad Sci U S A 112(29): 9040-9045.
  6. Holemans, T., Vandecaetsbeek, I., Wuytack, F. and Vangheluwe, P. (2014). Measuring Ca2+-dependent Ca2+-uptake activity in the mouse heart. Cold Spring Harb Protoc 2014(8): 876-886.
  7. Jidenko, M., Lenoir, G., Fuentes, J. M., M., le Maire and Jaxel, C. (2006). Expression in yeast and purification of a membrane protein, SERCA1a, using a biotinylated acceptor domain. Protein Expr Purif 48(1): 32-42.
  8. Kuhlbrandt, W. (2004). Biology, structure and mechanism of P-type ATPases. Nat Rev Mol Cell Biol 5(4): 282-295.
  9. Lin, C. H., Tan, E. K., Chen, M. L., Tan, L. C., Lim, H. Q., Chen, G. S. and Wu, R. M. (2008). Novel ATP13A2 variant associated with Parkinson disease in Taiwan and Singapore. Neurology 71(21): 1727-1732.
  10. Papadopulos, A., Vehring, S., Lopez-Montero, I., Kutschenko, L., Stockl, M., Devaux, P. F., Kozlov, M., Pomorski, T. and Herrmann, A. (2007). Flippase activity detected with unlabeled lipids by shape changes of giant unilamellar vesicles. J Biol Chem 282(21): 15559-15568.
  11. Park, J. S., Mehta, P., Cooper, A. A., Veivers, D., Heimbach, A., Stiller, B., Kubisch, C., Fung, V. S., Krainc, D., Mackay-Sim, A. and Sue, C. M. (2011). Pathogenic effects of novel mutations in the P-type ATPase ATP13A2(PARK9) causing Kufor-Rakeb syndrome, a form of early-onset parkinsonism. Hum Mutat 32(8): 956-964.
  12. Ramirez, A., Heimbach, A., Grundemann, J., Stiller, B., Hampshire, D., Cid, L. P., Goebel, I., Mubaidin, A. F., Wriekat, A. L., Roeper, J., Al-Din, A., Hillmer, A. M., Karsak, M., Liss, B., Woods, C. G., Behrens, M. I. and Kubisch, C. (2006). Hereditary parkinsonism with dementia is caused by mutations in ATP13A2, encoding a lysosomal type 5 P-type ATPase. Nat Genet 38(10): 1184-1191.
  13. Uemura, T. and Gerner, E. W. (2011). Polyamine transport systems in mammalian cells and tissues. Methods Mol Biol 720: 339-348.
  14. van Veen, S., Martin, S., Van den Haute, C., Benoy, V., Lyons, J., Vanhoutte, R., Kahler, J. P., Decuypere, J. P., Gelders, G., Lambie, E., Zielich, J., Swinnen, J. V., Annaert, W., Agostinis, P., Ghesquiere, B., Verhelst, S., Baekelandt, V., Eggermont, J. and Vangheluwe, P. (2020). ATP13A2 deficiency disrupts lysosomal polyamine export. Nature 578(7795): 419-424.

简介

[摘要] ATP13A2 / PARK9是一种晚期内/溶酶体P5B转运ATPase,与多种神经退行性疾病有关。我们最近将ATP13A2表征为溶酶体多胺出口者,这为未知的哺乳动物多胺转运系统的分子身份提供了线索。在这里,我们逐步描述了从过量表达人ATP13A2的酵母细胞中测量重组囊泡中放射性标记的多胺转运的方案。该方案是我们最新出版物的一部分(van Veen等,2020),将有助于表征其他假定的多胺转运蛋白的转运功能,例如P5B转运ATPase的同工型。


[背景] ATP13A2 / PARK9编码一种普遍表达的晚期内-/溶酶体膜蛋白,与一系列神经退行性疾病有关,例如早发性帕金森氏病(Di Fonzo等,2007 ;Lin等,2008)和Kufor -Rakeb综合征(伴痴呆的早期帕金森病)(Ramirez等,2006 ;Park等,2011)。ATP13A2属于P型转运ATPase ,是一类活性转运蛋白,由于ATP水解而暂时形成磷酸中间产物(Kuhlbrandt ,2004年)。ATP13A2是P5亚家族的成员,该家族已在20多年前通过基因组测序鉴定出来(Axelsen和Palmgren ,1998),并包含5种人同工型(ATP13A1-5)。然而,直到最近,当我们将ATP13A2表征为溶酶体多胺输出物时,ATP13A2的运输底物仍是未知的,其对精胺(SPM)表现出最高的亲和力(van Veen et al。,2020)。ATP13A2被两种调节性脂质磷脂酸和磷脂酰肌醇(3,5)双磷酸酯激活(Holemans等,2015 ; van Veen等,2020)。为了证明ATP13A2在膜上运输多胺,我们开发了一种运输试验,使用using标记的SPM([ 3 H] -SPM)和酵母膜衍生的囊泡重组表达具有C端BAD(生物素受体域)标签的人ATP13A2( ATP13A2-BAD)。

多胺转运分析始于在脂质磷脂酰胆碱和磷脂酸的存在下,由含有过量表达的ATP13A2-BAD的溶解酵母膜生产重组囊泡。生成的蛋白脂质体将同时包含从右向外和由内向外的重组ATP13A2(图1),并且我们利用这一原理建立了一个转运分析方法,其中我们跟踪了[ 3 H] -SPM在腔内的积累。囊泡。由于ATP13A2是溶酶体输出,因此应从内向外插入ATP13A2(面向内腔的胞质结构域),以允许[ 3 H] -SPM的腔积聚。在这种取向下,应在脂质体的内腔中补充ATP,以确保ATP结合和ATPase活性可以在核苷酸结合域发生。因此,我们在ATP和ATP再生系统(磷酸肌酸/肌酸磷酸激酶)的存在下重构蛋白脂质体。在从内向外的方向上,如果ATP存在于内部,则ATP13A2将促进蛋白脂质体中[ 3 H] -SPM的摄取,这与ATP13A2介导的多胺在细胞内从胞外质到胞质的转运是一致的(图1)。

图1.多胺转运分析背后原理的图形表示。为了测定多胺转运,我们使用脂质体小泡,由含有ATP13A2的酵母膜重构而成。生成的蛋白脂质体将同时包含右侧向外和内侧向外的重组ATP13A2。为使腔内[ 3 H] -SPM积累,应将ATP13A2内外插入(面向内腔的胞质结构域),因为ATP13A2是溶酶体输出物。另外,应像在P型ATPase中那样在蛋白脂质体的内腔中补充ATP ,ATP结合发生在位于胞质结构域之一的核苷酸结合位点。仅当蛋白脂质体内存在ATP时,[ 3 H] -SPM才在囊泡中积累,这与ATP13A2作为溶酶体多胺输出物的细胞作用一致。



与哺乳动物系统相比,我们的使用酵母衍生膜的方案为多胺转运分析提供了多个优势(例如,Uemura和Gerner ,2011)。尽管与哺乳动物细胞裂解相比,酵母粗提物的制备更加费力且费时,但好处在于酵母细胞生长快,易于培养,且成本低且生物量高。此外,酵母菌是易于延展的模型生物,易于遗传操作。此外,我们的协议代表了一种干净的体外技术,与细胞多胺摄取测定法相反。Ø乌尔多胺运输法适用于其他候选人胺转运体,这将有助于建立哺乳动物多胺运输系统的分子球员,这仍是未知。基于跨膜螺旋M4中底物结合域的高度保守性,其他哺乳动物P5B ATPase (ATP13A3-5)也很有可能在多胺转运系统中发挥作用,底物特异性,亚细胞定位可能略有不同和/或组织分布。因此,我们的转运分析对于表征其他相关P5B ATPase的转运功能也将是有价值的。

关键字:多胺, 精胺, 运输试验, 重构, 酵母细胞膜, P5 ATPase, ATP13A2

材料和试剂

笔记:
除非另有说明,否则所有材料和试剂均应保持在室温下。对于试剂的保存期限和存储温度,我们直接参考制造商的说明。
其他公司的等效材料和试剂也可能适用。

1.移液器技巧     
P10技巧(Sarstedt,目录号:70.1130.20)

P200技巧(萨尔特,目录号:70.760.102)

P1000提示(VWR,目录号:613-0738)

2.过滤移液器吸头     
P10过滤嘴(润滑脂,目录号:771288)

P200过滤嘴(润滑脂,目录号:739288)

P1000过滤嘴(润滑脂,目录号:740288)

3. Eppendorf管(Greiner,目录号:616201)     
4. Falcon管(格雷纳,catalo克数小号:188271 [ 15毫升] ,227261 [ 50毫升] )     
5.丁腈手套(VWR,目录号:112-2371)     
6.杜兰玻璃瓶(VWR,目录号小号:215-15 16 [ 500毫升] ,215-1517 [ 1 ,000毫升] )     
7.培养瓶(DWK生命科学,目录号:217715407)     
8.注射器驱动的过滤器单元(Merck,Millex ,目录号:SLGS033SB)     
9.培养皿(60毫米)(ThermoFisher ,目录号:123-17)     
10.瓶顶真空过滤系统(VWR,孔径为0.2 µm的完整过滤单元,目录号:514-0334) 
11.酸洗玻璃珠(Sigma-Aldrich,目录号:G8772) 
12.试管苏打玻璃(VWR,目录号:212-0013) 
13.膜过滤器,0.45微米孔径(Millipore公司,目录号:HAWP02500) 
14.酿酒酵母菌株W303-1B / Gal4-∆Pep4(leu2-3,his3-11,15,trp1-1 :: TRP1-GAL10-GAL4,ura3-1,ade2-1,canr ,cir +,∆Pep4 MATα) 
注意:S火车可应要求由相应的作者提供。

15.质粒DNA 
w ^ e,利用一个pYeDP60矢量(与2- μ米圆复制起点,URA3和ADE2选择性标记,和半乳糖诱导型启动子)含有人ATP13A2变体2的酵母密码子优化的cDNA版本随后是凝血酶切割位点和C -末端BAD标签(Jidenko等,2006; Azouaoui等,2014)(图2)。在我们的实验中,我们使用催化失活的ATP13A2变体,即E343A突变体(van Veen等,2020)作为阴性转运对照。E343位于去磷酸化的催化位点,在P型ATP酶(人ATP13A2同工型2中的341个TGES基序)中高度保守。

注:P lasmids请直接从相应的作者。




图2. ATP13A2表达质粒图的示意图。ATP13A2的表达质粒是修饰的pYeDP60质粒。所述pYeDP60载体含有2- μ米环复制起源,URA3和ADE2选择性标记,半乳糖诱导型GAL10 / CYC1启动子,多克隆位点,和PGK1终止子。将人ATP13A2基因克隆到具有凝血酶可裂解的BAD(生物素受体结构域)标签的质粒中(Jidenko等,2006 ;Azouaoui等,2014)。

16.粒状酵母提取物(默克,目录号:1.03753.0500) 
17.来自酪蛋白的蛋白ept (Tr yptone )(Merck,目录号:1.07213.2500) 
18. D-(+)-葡萄糖(Sigma-Aldrich,目录号:G8270) 
19. Cuvettes(VWR,目录号:634-0676) 
20. Tris基座(Sigma-Aldrich,Trizma基座,目录号:T1503) 
21.乙二胺四乙酸(EDTA)(BDH实验室耗材,目录号:280214S) 
22.盐酸(盐酸)发烟37%(Merck公司,目录号:1.00317.2501) 
23. HCl 0.5 N(Reagecon ,目录号:H20501) 
24.二水醋酸锂(LiAc )(Sigma-Aldrich,目录号:L6883) 
25.乙酸(西格玛奥德里奇,目录号:27225) 
26.鲱鱼精子的DNA(Sigma-Aldrich,目录号:D7290) 
27.聚乙二醇(PEG)(Sigma-Aldrich,目录号:P4338) 
28.二甲基亚砜(DMSO)(西格玛奥德里奇,目录号:276855) 
29.细菌用琼脂(BD,目录号:214010) 
30.不含尿嘧啶的酵母脱菌剂(Sigma-Aldrich,目录号:Y1501) 
31.不含氨基酸的酵母氮碱基(西格玛奥德里奇,目录号:Y0626) 
32.绝对乙醇(VWR,目录号:20821.296) 
33. D-(+)-半乳糖(Sigma-Aldrich,目录号:G0625) 
34. KCl (西格玛奥德里奇,目录号:P9541) 
35.山梨糖醇(Sigma-Aldrich,目录号:S1876) 
36. NaOH (Merck,目录号:649.1000) 
37. P henylmethylsulfonyl氟(PMSF)(Sigma-Aldrich公司,目录号:93482) 
38.蛋白酶抑制剂(Sigma-Aldrich,SIGMAFAST蛋白酶抑制剂鸡尾酒片剂,目录号:S8830) 
39. HEPES (西格玛奥德里奇,目录号:H3375) 
40.蔗糖(西格玛奥德里奇,目录号:S7903) 
41. CaCl 2 (西格玛奥德里奇,目录号:C3881) 
42.正十二烷基-β-D-麦芽吡喃糖苷(DDM)(Inalco ,目录号:1758-1350) 
43. Bradford试剂(Sigma-Aldrich,目录号:B6916) 
44. E gg磷脂酰胆碱(Avanti极性脂质,L-α-磷脂酰胆碱[鸡蛋,鸡肉] ,目录号:840051C) 
45. 18:1磷脂酸(Avanti极性脂质,1,2-二油酰基-sn-甘油-3-磷酸酯(钠盐),目录号:840875C) 
46. Bio-Beads SM-2吸附剂(BioRad ,目录号:1523920) 
47. ATP二钠盐(Roche Diagnostics,目录号:10127531001) 
48. MgCl 2六水合物(西格玛奥德里奇,目录号:M2670) 
49.磷酸肌酸二钠盐水合物(西格玛奥德里奇,目录号:P7936) 
50.肌酸磷酸激酶(Sigma-Aldrich,目录号:C3755) 
51.抗ATP13A2抗体(Sigma-Aldrich,目录号:A3361) 
52.抗兔IgG,HRP相连的抗体(Bioké ,目录号:7074S) 
53. γ-(N-吗啉代)丙磺酸(MOPS)(VWR,目录号:A1076.1000) 
54. KOH(Sigma-Aldrich,目录号:221473) 
55.二硫苏糖醇(DTT)(VWR,目录号:A2948.0025) 
56. SPM(Sigma-Aldrich,目录号:85590) 
57. [ 3 H] -SPM,1 mCi / ml,20 µM (美国放射性标记化学公司;目录号:ART0471) 
58.液体闪烁鸡尾酒(MP生物医学,Ecolite (+)TM液体闪烁液,目录号:882475) 
59.三氯甲烷库存(Sigma-Aldrich,目录号:C2432) 
60. 20%葡萄糖(请参阅食谱1) 
61. YPD琼脂平板(请参见配方2) 
62. YPD介质(请参见配方3) 
63. 10x TE股票(请参阅第4条) 
64. 10x LiAc库存(请参阅第5条) 
65. 1x TE / LiAc解决方案(请参阅第6条) 
66.单链鲱鱼精子DNA(请参见食谱7 ) 
67. 50%PEG w / v(请参阅第8章) 
68. PEG溶液(请参见配方9) 
69. SD-尿嘧啶琼脂平板(参见第10条) 
70. SD尿嘧啶培养基(请参见第11条) 
71. YPGE2x介质(请参见配方12) 
72. 20%半乳糖(请参阅食谱13) 
73. TEKS缓冲区(请参见配方14) 
74.缓冲区中的TES (请参见第15条) 
75. HS缓冲区(请参见配方16) 
76.缓冲区T(请参见第17条) 
77.缓冲区T / DDM(请参见配方18) 
78.缓冲液T /脂质混合物(请参见配方19) 
79. 0.1 M ATP(请参阅第20条) 
80. 0.1 M MgCl 2 (参见配方21) 
81. 0.2 M磷酸肌酸(见配方22) 
82. ATP再生系统(请参见第23条) 
83. 500 mM DTT(请参见配方24) 
84. 4x反应缓冲液(请参见配方25) 
85. 1x反应缓冲液(请参见配方26) 
86. 0.1 M MOPS(pH 7 .0 )(请参见配方27) 
87. 10 mM SPM(请参见配方28) 
88. 100 µl [ 3 H] -SPM /未标记的SPM混合物(请参见第29条) 

设备

移液器
P2移液器(Gilson,目录号:FA10001M)

P10移液器(Gilson,目录号:FA10002M)

P100移液器(Gilson,目录号:FA10004M)

P200移液器(Gilson,目录号:FA10005M)

P1000移液器(Gilson,目录号:FA10006M)

血清学吸移管(Sigma公司,BRAND ® ACCU -jet ®亲吸管控制器,目录号:Z637637)
血清移液器
5毫升(Sarstedt ,目录号:86.1253.001)

10毫升(Sarstedt ,目录号:86.1254.001)

25毫升(Sarstedt ,目录号:86.1685.001)

用于Ti45转子的超速离心管(Beckman Coulter,目录号:355655)
用于Ti70转子的超速离心管(Beckman Coulter,目录号:355630)
玻璃布氏过滤漏斗(Millipore公司,目录号:XX1014700)
磁力搅拌器(He idolph Instruments,MR Hei -Standard,目录号:505-20000-00)
带有摇床的冷藏培养箱(New Brunswick Scientific,型号:Innova 4230)
冰桶(例如,保丽龙箱)
高压灭菌器(LTE Scientific Ltd,100系列高压灭菌器)
M illiQ (M Q)水系统(Sartorius,Arium Pro)
分光光度计(贝克曼库尔特,型号:DU-640B)
带转子的离心机(Eppendorf,型号:5804 R )(Eppendorf,型号:A-4-44)
微量仪(Eppendorf,离心机5417 R)与转子(的Eppendorf,型号:F45-30-11)
涡流(VWR,型号:涡流-精灵® 2,目录号:444-5900)
Thermomixer(Eppendorf,型号:Thermomixer C omfort)
水浴(Memmert )
均质器(BioSpec产品,BeadBeater ,目录号:1107900EUR )
压力真空泵(Gelman Sciences,Gelman Little Giant,型号:13156)
汉密尔顿注射器(1毫升)(VWR,1001 LTN,货号:613-1300)
氮气排污系统(内部制造)
超速离心机(贝克曼库尔特公司,型号:Optima XPN-90)
超速离心机转子(贝克曼库尔特公司,型号:45 Ti )
超速离心机转子(贝克曼库尔特,型号:90 Ti )
头顶旋转器(Labinco BV,L28 Test-Tube Rotator,货号:28000)
真空过滤歧管(Millipore,目录号:XX2702550)
液体闪烁分析仪(Perkin Elmer,TRI-CARB 2900TR)

程序

酵母转化
我们转化牛逼他酵母ST雨W303-1B / GAL4-ΔPep4(leu2-3,his3-11,15,trp1-1 :: TRP1-GAL10-GAL4,ura3-1,ade2-1,canr ,CIR +, ΔPep4MATα),其中含有hATP13A2 WT或催化死亡的E343A突变体的pYeDP60载体,根据乙酸锂/单链载体DNA /聚乙二醇方法进行了较小的改动(Gietz和Woods,2002)。

压板从甘油原液对酵母一个YPD琼脂平板培养48小时,在30℃。
              从新鲜培养板上接种20 ml YPD培养基,并在30°C,230 rpm的振荡培养箱中孵育过夜。
在600 nm(OD 600 )下测量过夜培养物的OD 。
注意:1 OD 600大约等于10 7细胞/ ml。

在10 ml YPD中将培养物稀释至0.1 OD 600 ,并在30°C,230 rpm的振荡培养箱中孵育4-6小时,直到培养物的OD 600在0.4至0.8之间。
在摆动的水桶(700 xg ; 5分钟)中,将相当于2 OD 600 (=±2 x 10 7个细胞)的细胞量降低。
用1 ml无菌M Q洗涤沉淀,并用移液器吸头重悬。转移到Eppendorf管中。
使用微量离心机旋转细胞(2,500 xg ; 2分钟)。
将沉淀重悬于1 ml 1x TE / LiAc中。
使用微量离心机旋转细胞(2,500 xg ; 2分钟)。
将沉淀重悬于100 µl 1x TE / LiAc中。 
加入10微升单链鲱鱼精子DNA。
加入1-5 µg质粒DNA。
涡流。
加入600 µl PEG溶液。
涡流。
在30°C下孵育45分钟(体积=约730 µl)。
加入DMSO至终浓度为10%(约73 µl)。
在42°C下热震15分钟。
在冰上冷却1-2分钟。
使用微量离心机旋转细胞(2,500 xg ; 2分钟)。
将沉淀重悬于200 µl YPD中,并在30°C下孵育2小时。
使用微量离心机旋转细胞(2,500 xg ; 2分钟)。
将细胞沉淀重悬于无菌M Q中,然后平板接种至SD-尿嘧啶琼脂平板上。
在30℃至recove孵育48小时将板- [R转化体。

酵母菌
在这里,我们遵循了之前描述的类似策略(Jidenko等人,2006;Azouaoui等人,2014),但做了一些小的修改。

从新鲜培养皿中接种20 ml SD-尿嘧啶培养基(步骤A24),以进一步选择携带带有URA3选择标记的质粒的酵母细胞,并在振荡培养箱中于28°C和200 rpm孵育24 h。
测量培养物的OD 600 。
将100 ml SD-尿嘧啶培养基接种至最终OD 600为0.2,并在振荡培养箱中于28°C和200 rpm孵育12 h。
测量培养物的OD 600 。
将3.6 L的YPGE2X培养基接种至最终OD 600为0.05,并在振荡培养箱中于28°C和175 rpm孵育36 h。
注意:九1 ,装有400 ml酵母培养物000毫升培养瓶中。

用2%半乳糖(每500 ml培养物中加入50 ml 20%半乳糖溶液50 ml)诱导ATP13A2-BAD表达,并在振荡培养箱中于18°C和175 rpm孵育12 h。
注意:Ť他温度降档从28℃至18℃下会降低蛋白质合成速率,因此,有利于适当的蛋白折叠,增加正确折叠和功能的过量表达的蛋白质的产率。另外,较低的温度将减少蛋白质降解。

重复半乳糖诱导,以确保高水平的ATP13A2-BAD过表达,并在振荡培养箱中于18°C和175 rpm孵育另外12 h。
旋转酵母细胞(1,000 xg ; 10分钟; 4°C)。
称量沉淀。
注意:产量通常为20-30 g / L酵母培养物。细胞沉淀可以直接处理或在-20°C下保存最多2周。

酵母膜制备
如果将酵母细胞沉淀物冷冻,则在冰上解冻。
将细胞沉淀重悬于TEKS缓冲液中(体积(ml)=步骤2中确定的细胞沉淀重量(g)的2倍),并在4°C下孵育15分钟,同时与磁力搅拌器混合。
旋转细胞(1,000 xg ; 10分钟; 4°C)。
将沉淀重悬于TES缓冲液中(体积(ml)=大约1倍于细胞沉淀的重量(g),如S b步B9中所确定)。
使用BeadBeater破坏酵母细胞。
用200 ml酵母悬浮液填充室(如果不够,则在缓冲液中添加TES )并添加200 ml冷玻璃珠。分庭应尽可能满。
将碎冰和水装满冰水套。
在冷室中搅拌5分钟。每隔一分钟暂停3分钟,以减少样品和BeadBeater的加热。
使用布氏漏斗和真空泵回收粗提物。
使用pH纸测试裂解物的pH,必要时用饱和NaOH溶液将粗提液的pH调节至7.5 。
将粗提取物以2,000 xg离心20分钟(4°C)。
使用45 Ti型超速离心转子(4°C)将得到的上清液以20,000 xg离心20分钟。
使用45 Ti型超速离心转子(4°C)以200,000 xg离心所得上清液1 h 。
将所得沉淀物(即,轻膜级分P3)重悬于HS缓冲液中。
注意:重悬液的体积应由肉眼确定,应为均匀溶解沉淀物所需的最小量。

使用经典的Bradford测定法(以已知的BSA浓度作为蛋白质标准品)确定P3馏分的蛋白质浓度。
注意:测得的蛋白质浓度通常在35-50 mg / ml之间。为了进行下游重组,每个实验要求的最低总产量为70 mg。

分装每2 ml P3膜,并在液体N 2中冷冻。
储存在-80°C。
注意:膜可以在-80°C下保存最多6个月。

酵母膜的重构
Ť ø重新构建酵母膜,我们遵循类似的策略如前描述(帕帕多普洛斯等人,2007)有一些修改。

在缓冲液T / DDM中将2 ml P3膜(在冰上融化)稀释至10 µg / µl。
注意:总蛋白质量应至少为70毫克。

在头顶旋转器中于4°C孵育45分钟。
使用90 Ti型超速离心转子(4°C)以200,000 xg离心30分钟,使不溶性部分沉淀。
补充2毫升上清液,即,德TERGENT提取物,用等体积的缓冲液的T /脂质混合和条件“内部ATP”,也ATP再生系统的等体积的(表1) :

表1.重组样品的准备

健康)状况

“没有ATP”

“ ATP内部”

洗涤剂提取物

2毫升

2米升

缓冲液T /脂质混合

2毫升

2毫升

ATP再生系统

--

2毫升

在室温下,将样品与100 mg / ml Bio-Beads在头顶旋转器中孵育3小时,以去除DDM并重组蛋白脂质体。
加入200 mg / ml Bio-Beads,在头顶旋转器中于4°C孵育过夜。
使用90 Ti型超速离心转子(4°C)以200,000 xg离心1 h 。
通过将沉淀重悬于2 ml缓冲液T中来回收囊泡。
注意:测得的蛋白质浓度通常在1-5 mg / ml之间。

使用经典的Bradford测定法确定蛋白质浓度。
通过蛋白质印迹检查重构的蛋白质水平(图3)。



图3.酵母膜和重组囊泡的免疫印迹。含有ATP13A2-BAD的P3膜被去污剂DDM溶解。去污剂提取物补充有脂质磷脂酰胆碱和磷脂酸,然后用Bio-Beads处理以去除DDM并重组蛋白脂质体(“无ATP”)。为了产生包含腔内ATP(“ ATP内部”)的蛋白脂质体,我们在与Bio-Beads一起孵育之前添加了ATP和ATP再生系统。使用一抗ATP13A2一抗(1 / 1,000)和缀合HRP的二抗(1 / 2,000)通过Western印迹分析检查重构的ATP13A2-BAD蛋白水平。每个泳道上样12 µl样品。

多胺转运分析
注意:多胺运输测定应由授权人员在指定的放射性区域内进行,并且必须采取放射性物质处理预防措施。应始终佩戴手套,实验室护目镜,实验室外套和用于监测个人暴露的剂量计。该工作应在有机玻璃滤网后面进行,并且只能使用带有过滤器的移液器吸头。放射性物质的存储以及固体和液体放射性废物的处置均应遵守机构的规定。
注意:在60分钟内测量[ 3 H] -SPM对新鲜制备的囊泡的吸收。

在缓冲液T中将新鲜制备的囊泡(“无ATP”或“ ATP内部”)稀释至1 µg / µl。
注意:我们通常使用1毫升的囊泡。至少需要300 µg(“ ATP内在”囊泡)– 600 µg(“无ATP”内囊泡)才能进行下一步。

将四个0.45 µm Millipore过滤器放在过滤歧管上(图4)。
注意:过滤器在M Q中是预湿的。使用镊子固定住它们。

用4 ml 1x反应缓冲液洗涤每个过滤器。
在不同条件下(在duplo中)将以下物质移入反应管中(表2):



表2.用于运输测定的样品制备

健康)状况

“没有ATP”

“ ATP不在一边”

“ ATP在身边”

4x反应缓冲液

250微升

250微升

250微升

“无ATP”囊泡

150微升

150微升

--

“ ATP内在”囊泡

--

--

150微升

ATP再生系统

--

--

200微升

质量Q

500微升

500微升

300微升

轻轻涡旋以彻底混合(避免形成气泡)。
将反应管置于37°C水浴中5分钟。
加入100 µl [ 3 H] -SPM /未标记的SPM混合物(最终总SPM浓度:1 mM )开始反应。
在选定的时间点(0分钟,10分钟)取出300微升(45微克囊泡),然后将每个等分试样吸移到不同的过滤器上,以除去液相。
清洗每个滤波器用4ml 1X REA ction缓冲器。             
将过滤器放入装有7毫升闪烁液的闪烁瓶中。
对其余的反应管重复步骤。
用液体闪烁计数器测量样品。



图4.用于多胺转运测定的实验装置。过滤歧管(1)连接到带有用于放射性废物(3)的收集器的真空泵(2 )。水浴(4)。

数据分析

所述的多胺转运测定法测量了孵育10分钟后[ 3 H] -SPM在含有由内而外ATP13A2和管腔内ATP(“ ATP内在”状态)的重构小泡中的积累。在不同的控制条件下并行进行实验。脂蛋白体与过表达的ATP13A2仅包含腔外ATP(“ATP外”状态),或在所有(“无ATP”条件)不具有ATP作为阴性对照,因为这些小泡不占用[ 3 H] -SPM由于缺乏ATP结合在由内而外重构的ATP13A2蛋白的腔核苷酸结合域上(图1)。此外,包含过表达的无催化活性的ATP13A2变体(E343A)的重构囊泡作为阴性转运对照。通过闪烁计数定量[ 3 H] -SPM摄取,并将每种情况的值归一化为0分钟。对于每个摄取时间,进行重复测定。代表性图如图5所示。




图5.多胺转运分析。相对于催化死亡的突变体E343A(阴性对照),在ATP13A2 WT过表达的酵母膜来源的囊泡中摄取[ 3 H] -SPM 。条件“无ATP”代表没有ATP的重组囊泡,而条件“外部ATP”和“内部ATP”分别代表包含脂质外或腔内ATP和ATP再生系统的蛋白脂质体(van Veen等人,2020) 。

笔记

可以将此处描述的协议扩展为执行以下类型的测量:
1.时间依赖性应该是线性的,它可以在不同时间点在0的范围内进行验证- 30分钟。吸收率可以从斜率计算。           
2.通过测试一系列总SPM浓度(感冒和[ 3 H] -SPM在一起标记的浓度)在0的生理范围内,可以生成剂量/反应曲线。0 1 µM - 10 mM。浓度依赖性允许确定对SPM的表观亲和力(K m )和转运蛋白的最大周转率(V max )。动力学参数是通过Hill方程通过非线性回归分析计算得出的,如Holemans等人所述。(2014年)。           

菜谱

20%葡萄糖
将500 ml M Q放入带搅拌棒的大烧杯中
加200克葡萄糖
搅拌几分钟
最终化体积为1 ,000毫升与中号Q
过滤消毒溶液
YPD琼脂平板
注:P酸酯应储存在4℃下在黑暗中。

将3 g酵母提取物,6 g蛋白p和6 g琼脂放在Duran玻璃瓶中
添加M Q至270毫升
在121 °C高压灭菌至少30分钟
加入30毫升20%葡萄糖
倒入培养皿
YPD培养基
将10克酵母提取物和20克蛋白ept放入杜兰玻璃瓶中
添加M Q至900毫升
高压釜 121 °C至少30分钟
加入100毫升20%葡萄糖
10x TE储备液(0.1 M Tris - HCl ,pH 7.5,0.01 M EDTA)
0.606克Tris

0.146克EDTA

添加M Q至50毫升

用HCl调节pH值至7.5

过滤消毒溶液

10x LiAc库存(1 M LiAc ,pH 7.5)
5.101克醋酸锂

添加M Q至50毫升

用稀乙酸调节pH至7.5

过滤消毒溶液

1x TE / LiAc解决方案
将120 µl 10x TE储备液,120 µl 10x LiAc储备液和900 µl无菌M Q混合在一起

单链鲱鱼精子DNA
DNA在水浴中煮沸20分钟,然后立即在冰上冷却

50%聚乙二醇w / v
称出25 g PEG
加入无菌的M Q至50毫升
过滤消毒溶液
PEG溶液(40%PEG,1x TE,1x LiAc )
将800 µl 50%PEG,100 µl 10x TE储备液和100 µl 10x LiAc储备液混合在一起

SD-尿嘧啶琼脂平板
注:P酸酯应储存在4℃下在黑暗中。

将5克琼脂,0.475克不含尿嘧啶的酵母菌落混合物和1.675克不含氨基酸的酵母氮碱基放入杜兰玻璃瓶中
添加M Q至200毫升
在121 °C高压灭菌至少30分钟
加50毫升20%葡萄糖
倒入培养皿
SD尿嘧啶培养基
注:中号edium应避光保存。

将0.95 g不含尿嘧啶的酵母菌落混合物和3.35 g不含氨基酸的酵母氮碱基放入Duran玻璃瓶中
添加M Q至450毫升
在121 °C高压灭菌至少30分钟
加50毫升20%葡萄糖
YPGE 2x中
将20克酵母提取物和20克蛋白ept放入杜兰玻璃瓶中
添加M Q至725毫升
在121 °C高压灭菌至少30分钟
加50毫升20%葡萄糖
加入27毫升乙醇
20%半乳糖
将500 ml M Q放入带搅拌棒的大烧杯中
加入200克半乳糖
混合时加热,使其溶解
最终化体积为1 ,000毫升与中号Q
过滤消毒溶液
TEKS缓冲液(50 mM Tris-HCl ,pH 7.5、1 mM EDTA,0.1 M KCl,0.6 M山梨糖醇) 
注意:乙uffer应保存在4℃。

6.057克Tris

0.292克EDTA

7.456克KCl

109.302 g s轨道醇

添加M Q至1000毫升

用HCl调节pH值至7.5

TES在缓冲液中(50 mM Tris-HCl ,pH 7.5、1 mM EDTA,0.6 M山梨糖醇,1 mM PMSF,蛋白酶抑制剂混合物)              
注意:乙uffer应保存在4℃。

6.057克Tris

0.292克EDTA

              109.302克山梨糖醇

添加中号Q上为1 ,000毫升

用HCl调节pH值至7.5

在实验当天添加Sigma Fast蛋白酶抑制剂混合物。对于50毫升缓冲液,加1片

PMSF是在使用前添加的。对于50 ml的缓冲液,添加500 µl 100 mM PMSF

HS缓冲液(20 mM HEPES - Tris ,pH 7.4,0.3 M蔗糖,0.1 mM CaCl 2 )
注意:乙uffer应保存在4℃。

0.477克HEPES

10.269克蔗糖

0.0015克CaCl 2

添加M Q至100毫升

用Tris将pH调节至7.4

缓冲液T(10 mM Tris-HCl ,pH 7.4和1 mM EDTA)
注意:乙uffer应保存在4℃。

0.121克Tris

0.029克EDTA

添加M Q至100毫升

用HCl调节pH值至7.4

缓冲液T / DDM(缓冲液T补充有1.4%DDM w / v)
20 ml缓冲液T

280毫克DDM

缓冲液T /脂质混合物(缓冲液T含有0.7%DDM w / v,4.5 mM卵磷脂酰胆碱和0.5 mM 18:1磷脂酸(PA)
将173 mg卵磷脂酰胆碱(6.92 ml的25 mg / ml氯仿原液)加入玻璃管中,并在氮气流下干燥以制成脂质膜
将18 mg 18:1 PA(1.8 ml的10 mg / ml氯仿原液)添加到脂质膜中并在氮气流下干燥
在补充了0.35 g DDM的50 ml缓冲液T中溶解脂膜
ATP 0.1 M
ATP 0.551克

添加M Q至10毫升

分装并储存在-20 °C

0.1 M氯化镁2
0.203克氯化镁2

添加M Q至10毫升

0.2 M磷酸肌酸
1克磷酸肌酸

添加M Q至19.6 ml

分装并储存在-20 °C

ATP再生系统
200微升0.1 M ATP

200微升0.1 M MgCl 2

200 µl 0.2 M磷酸肌酸

200 µl肌酸磷酸激酶(1 U / µl)

1.2毫升M Q

500毫米DTT
0.771克DTT

添加M Q至10毫升

分装并储存在-20°C

4x反应缓冲液(200 mM MOPS,400 mM KCl ,44 mM MgCl 2和4 mM DTT)
注意:乙uffer应保存在4℃。

4.184克MOPS

2.982克氯化钾

0.894克MgCl 2

添加M Q至100毫升

用KOH调节pH到7.0

在使用前添加DTT。对于5 ml缓冲液,添加40 µl 500 mM储备液

1x反应缓冲液(50 mM MOPS,100 mM KCl ,11 mM MgCl 2和1 mM DTT)
100 ml 4x反应缓冲液

300毫升M Q

0.1 M MOPS(pH 7 .0 )
2.093克MOPS

添加M Q至100毫升

用KOH调节pH到7.0

10 mM SPM
0.020克SPM

加入0.1 M MOPS(pH 7 .0 )至10 ml

分装并储存在-80 °C的氮气下。解冻后的等分试样将不再使用

[ 3 H] -SPM /未标记SPM混合物(10 µCi,0.2 µM [ 3 H] -SPM,10 mM SPM,总SPM浓度:9.9 mM )
10 µl [ 3 H] -SPM

990微升10 mM SPM

致谢

所描述的协议最初是在van Veen等人的方法中发布的。,2020年。这项工作由Fonds Wetenschappelijk Onderzoek (FWO,法兰德斯研究基金会)(G094219N,SBO Neuro-TRAFFIC S006617N和1503117N),KU鲁汶大学(LysoCaN C16 / 15/073 )和伊丽莎白女王医学科学基金会资助。 ,Valine de Spoelberch奖。SVV 。是一位有抱负的FWO研究员(11Y7518N)。我们感谢奥尔胡斯大学的J. Lyons博士在生成ATP13A2 BAD标签融合构建体方面的帮助。

利益争夺

作者声明没有任何金融或非金融竞争利益。

参考文献

              Axelsen,KB和Palmgren,MG(1998)。P型ATPase超家族中底物特异性的演变。分子进化杂志46(1):84-101。              
              Azouaoui,H.,Montigny,C.,Ash,MR,Fijalkowski,F.,Jacquot,A.,Gronberg,C.,Lopez-Marques,RL,Palmgren,MG,Garrigos,M.,le Maire,M., Decottignies,P.,Gourdon,P.,Nissen,P.,Champeil,P.和Lenoir,G.(2014年)。一种高产量的共表达系统,用于纯化完整的Drs2p-Cdc50p脂质翻转酶复合物,该复合物严重依赖于磷脂酰肌醇4-磷酸酯并由其稳定。PLoS One 9(11):e112176。
              Di Fonzo,A.,Chien,HF,Socal,M.,Giraudo,S.,Tassorelli,C.,Iliceto,G.,Fabbrini,G.,Marconi,R.,Fincati,E.,Abbruzzese,G., Marini,P.,Squitieri,F.,Horstink,MW,Montagna,P.,Libera,AD,Stocchi,F.,Goldwurm,S.,Ferreira,JJ,Meco,G.,Martignoni,E.,Lopiano,L ,Jardim,LB,Oostra,BA,Barbosa,ER,Italian Parkinson Genetics,N。和Bonifati,V。(2007)。少年帕金森病和年轻的帕金森病发病中的ATP13A2错义突变。神经病学68(19):1557-1562。              
Gietz,RD和Woods,RA(2002)。通过乙酸锂/单链载体DNA /聚乙二醇法转化酵母。方法酶350:87-96。
              霍尔曼斯(Tholes),索伦森(DM),范·文(Van Veen),S。,马丁·S。,赫曼斯(M.D.),凯默(Kemmer),GC,范登豪特(C.) 。,Wuytack,F.,Palmgren,M.,Eggermont,J.和Vangheluwe,P.(2015)。脂质开关可解除与帕金森氏病相关的ATP13A2。美国国家科学院院刊112(29):9040-9045。
              Holemans,T.,Vandecaetsbeek,I.,Wuytack,F.和Vangheluwe,P.(2014)。测量小鼠心脏中Ca 2+依赖性Ca 2+摄取活性。Cold Spring Harb Protoc 2014(8):876-886。
              Jidenko,M.,Lenoir,G.,Fuentes,JM,Le Maire,M.和Jaxel,C.(2006)。使用生物素化的受体域在酵母中表达和纯化膜蛋白SERCA1a。Protein Expr Purif 48(1):32-42。              
              Kuhlbrandt,W.(2004)。P型ATP酶的生物学,结构和机制。Nat Rev Mol Cell Biol 5(4):282-295。              
              Lin CH,Tan,EK,Chen,ML,Tan,LC,Lim,HQ,Chen,GS and Wu,RM(2008)。台湾和新加坡与帕金森病有关的新型ATP13A2变异体。神经病学71(21):1727-1732。
              Papadopulos,A.,Vehring,S.,Lopez-Montero,I.,Kutschenko,L.,Stockl,M.,Devaux,PF,Kozlov,M.,Pomorski,T. and Herrmann,A.(2007年)。通过巨大的单层囊泡形状变化,未标记脂质检测到的脂酶活性。生物化学杂志282(21):15559-15568。
              Park,JS,Mehta,P.,Cooper,AA,Veivers,D.,Heimbach,A.,Stiller,B.,Kubisch,C.,Fung,VS,Krainc,D.,Mackay-Sim,A.和Sue ,CM(2011)。P型ATPase ATP13A2(PARK9)中新突变的致病作用,导致Kufor-Rakeb综合征,这是一种早期发作的帕金森病。嗡嗡声突变体32(8):956-964。              
拉米雷斯,A。,海姆巴赫,A。格伦德曼,J。,斯蒂勒,B。汉普郡,D。,西德,LP,戈贝尔,I。,穆拜丁,AF,Wriekat,AL,罗珀,J。,艾丁,A.,Hillmer,AM,Karsak,M.,Liss,B.,Woods,CG,Behrens,MI和Kubisch,C。(2006)。遗传性帕金森氏症和痴呆症是由ATP13A2突变引起的,该突变编码溶酶体5 P型ATP酶。Nat Genet 38(10):1184-1191。              
Uemura,T.和Gerner,EW(2011)。哺乳动物细胞和组织中的多胺转运系统。方法分子生物学720:339-348。
范·范·南(Van Veen,S.),马丁·南(Martin,S.),范登·豪特(Van den Haute,C.),贝诺伊(Benoy,V.),里昂(Lyons),J.,范豪特(Vanhoutte),R. ,Zielich,J.,Swinnen,JV,Annaert,W.,Agostinis,P.,Ghesquiere,B.,Verhelst,S.,Baekelandt,V.,Eggermont,J。和Vangheluwe,P。(2020)。ATP13A2缺乏会干扰溶酶体多胺输出。自然578(7795):419-424。
登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Veen, S. V., Martin, S., Schuermans, M. and Vangheluwe, P. (2021). Polyamine Transport Assay Using Reconstituted Yeast Membranes. Bio-protocol 11(2): e3888. DOI: 10.21769/BioProtoc.3888.
提问与回复
提交问题/评论即表示您同意遵守我们的服务条款。如果您发现恶意或不符合我们的条款的言论,请联系我们:eb@bio-protocol.org。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。