-
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)
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Filter pipette tips
P10 filter tips (Greiner, catalog number: 771288)
P200 filter tips (Greiner, catalog number: 739288)
P1000 filter tips (Greiner, catalog number: 740288)
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Eppendorf tubes (Greiner, catalog number: 616201)
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Falcon tubes (Greiner, catalog numbers: 188271 [15 ml], 227261 [50 ml])
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Nitrile gloves (VWR, catalog number: 112-2371)
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Duran glass bottle (VWR, catalog numbers: 215-1516 [500 ml], 215-1517 [1,000 ml])
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Culture flasks (DWK Life Sciences, catalog number: 217715407)
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Syringe-driven filter unit (Merck, Millex, catalog number: SLGS033SB)
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Petri dishes (60 mm) (ThermoFisher, catalog number: 123-17)
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Bottle top vacuum filtration system (VWR, complete filtration unit with 0.2 µm pore size, catalog number: 514-0334)
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Acid-washed glass beads (Sigma-Aldrich, catalog number: G8772)
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Test tubes Soda glass (VWR, catalog number: 212-0013)
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Membrane filters, 0.45 μm pore size (Millipore, catalog number: HAWP02500)
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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.
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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).
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Yeast extract granulated (Merck, catalog number: 1.03753.0500)
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Peptone from casein (Tryptone) (Merck, catalog number: 1.07213.2500)
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D-(+)-Glucose (Sigma-Aldrich, catalog number: G8270)
-
Cuvettes (VWR, catalog number: 634-0676)
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Tris base (Sigma-Aldrich, Trizma base, catalog number: T1503)
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Ethylenediaminetetra-acetic acid (EDTA) (BDH Laboratory Supplies, catalog number: 280214S)
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Hydrochloric acid (HCl) fuming 37% (Merck, catalog number: 1.00317.2501)
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HCl 0.5 N (Reagecon, catalog number: H20501)
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Lithium acetate dihydrate (LiAc) (Sigma-Aldrich, catalog number: L6883)
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Acetic acid (Sigma-Aldrich, catalog number: 27225)
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DNA from herring sperm (Sigma-Aldrich, catalog number: D7290)
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Polyethylene glycol (PEG) (Sigma-Aldrich, catalog number: P4338)
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Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855)
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Bacto Agar (BD, catalog number: 214010)
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Yeast dropout mix without uracil (Sigma-Aldrich, catalog number: Y1501)
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Yeast nitrogen base without amino acids (Sigma-Aldrich, catalog number: Y0626)
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Ethanol absolute (VWR, catalog number: 20821.296)
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D-(+)-Galactose (Sigma-Aldrich, catalog number: G0625)
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KCl (Sigma-Aldrich, catalog number: P9541)
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Sorbitol (Sigma-Aldrich, catalog number: S1876)
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NaOH (Merck, catalog number: 6498.1000)
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Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 93482)
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Protease inhibitor (Sigma-Aldrich, SIGMAFAST Protease Inhibitor Cocktail Tablets, catalog number: S8830)
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HEPES (Sigma-Aldrich, catalog number: H3375)
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Sucrose (Sigma-Aldrich, catalog number: S7903)
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CaCl2 (Sigma-Aldrich, catalog number: C3881)
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n-Dodecyl-β-D-Maltopyranoside (DDM) (Inalco, catalog number: 1758-1350)
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Bradford reagent (Sigma-Aldrich, catalog number: B6916)
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Egg phosphatidylcholine (Avanti Polar Lipids, L-α-phosphatidylcholine [Egg, Chicken], catalog number: 840051C)
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18:1 phosphatidic acid (Avanti Polar Lipids, 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt), catalog number: 840875C)
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Bio-Beads SM-2 Adsorbents (BioRad, catalog number: 1523920)
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ATP disodium salt (Roche Diagnostics, catalog number: 10127531001)
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MgCl2 hexahydrate (Sigma-Aldrich, catalog number: M2670)
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Phosphocreatine disodium salt hydrate (Sigma-Aldrich, catalog number: P7936)
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Creatine phosphokinase (Sigma-Aldrich, catalog number: C3755)
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Anti-ATP13A2 antibody (Sigma-Aldrich, catalog number: A3361)
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Anti-rabbit IgG, HRP-linked antibody (Bioké, catalog number: 7074S)
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γ-(N-Morpholino)propanesulphonic acid (MOPS) (VWR, catalog number: A1076.1000)
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KOH (Sigma-Aldrich, catalog number: 221473)
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Dithiothreitol (DTT) (VWR, catalog number: A2948.0025)
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SPM (Sigma-Aldrich, catalog number: 85590)
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[3H]-SPM, 1 mCi/ml, 20 µM (American Radiolabeled Chemicals, Inc.; catalog number: ART0471)
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Liquid scintillation cocktail (MP Biomedicals, Ecolite(+)TM Liquid Scintillation Fluid, catalog number: 882475)
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Chloroform stock (Sigma-Aldrich, catalog number: C2432)
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20% glucose (see Recipe 1)
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YPD-agar plates (see Recipe 2)
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YPD medium (see Recipe 3)
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10x TE stock (see Recipe 4)
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10x LiAc stock (see Recipe 5)
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1x TE/LiAc solution (see Recipe 6)
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Single-stranded herring sperm DNA (see Recipe 7)
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50% PEG w/v (see Recipe 8)
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PEG solution (see Recipe 9)
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SD-uracil agar plates (see Recipe 10)
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SD-uracil medium (see Recipe 11)
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YPGE2x medium (see Recipe 12)
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20% galactose (see Recipe 13)
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TEKS buffer (see Recipe 14)
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TESin buffer (see Recipe 15)
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HS buffer (see Recipe 16)
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Buffer T (see Recipe 17)
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Buffer T/DDM (see Recipe 18)
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Buffer T/lipid mix (see Recipe 19)
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0.1 M ATP (see Recipe 20)
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0.1 M MgCl2 (see Recipe 21)
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0.2 M phosphocreatine (see Recipe 22)
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ATP-regenerating system (see Recipe 23)
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500 mM DTT (see Recipe 24)
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4x reaction buffer (see Recipe 25)
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1x reaction buffer (see Recipe 26)
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0.1 M MOPS (pH 7.0) (see Recipe 27)
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10 mM SPM (see Recipe 28)
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100 µl [3H]-SPM/unlabeled SPM mix (see Recipe 29)
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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)
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Serological pipettor (Sigma, BRAND® accu-jet® pro pipette controller, catalog number: Z637637)
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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)
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Ultracentrifuge tubes for Ti45 rotor (Beckman Coulter, catalog number: 355655)
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Ultracentrifuge tubes for Ti70 rotor (Beckman Coulter, catalog number: 355630)
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Glass Büchner filter funnel (Millipore, catalog number: XX1014700)
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Magnetic stirrer (Heidolph Instruments, MR Hei-Standard, catalog number: 505-20000-00)
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Refrigerated incubator with shaker (New Brunswick Scientific, model: Innova 4230)
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Ice bucket (e.g., styrofoam box)
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Autoclave (LTE Scientific Ltd, Series 100 Autoclave)
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MilliQ (MQ) water system (Sartorius, Arium Pro)
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Spectrophotometer (Beckman Coulter, model number: DU-640B)
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Centrifuge (Eppendorf, model: 5804 R) with rotor (Eppendorf, model: A-4-44)
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Microcentrifuge (Eppendorf, Centrifuge 5417 R) with rotor (Eppendorf, model: F45-30-11)
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Vortex (VWR, model: Vortex-Genie® 2, catalog number: 444-5900)
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Thermomixer (Eppendorf, model: Thermomixer Comfort)
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Water bath (Memmert)
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Homogenizer (BioSpec products, BeadBeater, catalog number: 1107900EUR)
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Pressure vacuum pump (Gelman Sciences, Gelman Little Giant, model: 13156)
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Hamilton syringes (1 ml) (VWR, 1001 LTN, catalog number: 613-1300)
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Nitrogen gas blow-down system (made in-house)
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Ultracentrifuge (Beckman Coulter, model: Optima XPN-90)
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Ultracentrifuge rotor (Beckman Coulter, model: Type 45 Ti)
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Ultracentrifuge rotor (Beckman Coulter, model: Type 90 Ti)
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Head-over-head rotator (Labinco BV, L28 Test-Tube Rotator, catalog number: 28000)
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Vacuum filtration manifold (Millipore, catalog number: XX2702550)
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Liquid scintillation analyzer (Perkin Elmer, TRI-CARB 2900TR)
The here-described protocol can be expanded to perform the following types of measurements:
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20% glucose
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Put 500 ml MQ in a large beaker glass with a stir bar
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Add 200 g glucose
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Stir the solution for a few minutes
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Finalize volume to 1,000 ml with MQ
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Filter sterilize the solution
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YPD-agar plates
Note: Plates should be stored at 4 °C in the dark.
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Put 3 g yeast extract, 6 g peptone and 6 g agar in a Duran glass bottle
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Add MQ up to 270 ml
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Autoclave at 121 °C for at least 30 min
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Add 30 ml 20% glucose
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Pour into Petri dishes
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YPD medium
-
Put 10 g yeast extract and 20 g peptone in a Duran glass bottle
-
Add MQ up to 900 ml
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Autoclave at 121 °C for at least 30 min
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Add 100 ml 20% glucose
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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
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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
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1x TE/LiAc solution
Mix together 120 µl 10x TE stock, 120 µl 10x LiAc stock and 900 µl sterile MQ
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Single-stranded herring sperm DNA
DNA is boiled for 20 min in water bath and then immediately cooled on ice
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50% PEG w/v
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Weigh off 25 g PEG
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Add sterile MQ up to 50 ml
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Filter sterilize the solution
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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
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SD-uracil agar plates
Note: Plates should be stored at 4 °C in the dark.
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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
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Add MQ up to 200 ml
-
Autoclave at 121 °C for at least 30 min
-
Add 50 ml 20% glucose
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Pour into Petri dishes
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SD-uracil medium
Note: Medium should be stored in the dark.
-
Put 0.95 g yeast dropout mix without uracil and 3.35 g yeast nitrogen base without amino acids in a Duran glass bottle
-
Add MQ up to 450 ml
-
Autoclave at 121 °C for at least 30 min
-
Add 50 ml 20% glucose
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YPGE 2x medium
-
Put 20 g yeast extract and 20 g peptone in a Duran glass bottle
-
Add MQ up to 725 ml
-
Autoclave at 121 °C for at least 30 min
-
Add 50 ml 20% glucose
-
Add 27 ml ethanol
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20% galactose
-
Put 500 ml MQ in a large beaker glass with a stir bar
-
Add 200 g galactose
-
Heat up while mixing to get it into solution
-
Finalize volume to 1,000 ml with MQ
-
Filter sterilize the solution
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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
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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
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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
-
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
-
Buffer T/DDM (buffer T supplemented with 1.4% DDM w/v)
20 ml buffer T
280 mg DDM
-
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)
-
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
-
Add 18 mg 18:1 PA (1.8 ml of 10 mg/ml chloroform stock) to the lipid film and dry under nitrogen stream
-
Resolubilize the lipid film in 50 ml buffer T supplemented with 0.35 g DDM
-
0.1 M ATP
0.551 g ATP
Add MQ up to 10 ml
Aliquot and store at -20 °C
-
0.1 M MgCl2
0.203 g MgCl2
Add MQ up to 10 ml
-
0.2 M phosphocreatine
1 g phosphocreatine
Add MQ up to 19.6 ml
Aliquot and store at -20 °C
-
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
-
500 mM DTT
0.771 g DTT
Add MQ up to 10 ml
Aliquot and store at -20 °C
-
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
-
1x reaction buffer (50 mM MOPS, 100 mM KCl, 11 mM MgCl2, 1 mM DTT)
100 ml 4x reaction buffer
300 ml MQ
-
0.1 M MOPS (pH 7.0)
2.093 g MOPS
Add MQ up to 100 ml
Adjust pH to 7.0 with KOH
-
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
-
[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
The authors declare no financial or non-financial competing interests.