Radio labeled PAPS ([³⁵S] PAPS:1.66Ci/mmol)

Zymolyase (Zymolyase-100T; Seikagaku Biobusiness, Tokyo, Japan)

Protease inhibitors I (5 µg/mL of pepstatin A and 1 mM of phenylmethylsulfonyl fluoride)

Protease inhibitors II (1 mM of phenylmethylsulfonyl fluoride, 1 µg/mL of leupeptin, 1 µg/mL of aprotinin, and 1 µg/mL of pepstatin A)

Synthetic defined medium　(1–3)

Spheroplast buffer (1.4 M sorbitol, 50 mM of potassium phosphate, pH 7.5, 10 mM of NaN₃, 40 mM of 2-mercaptoethanol, and 1 mg of Zymolyase 100T/g wet cells)

Wash buffer (1.0 M sorbitol)

Lysis buffer I (0.8 M sorbitol, 10 mM of triethanolamine, pH7.2, and protease inhibitors I)

Lysis buffer II (10 mM of HEPES-Tris, pH 7.4, 0.25 M sucrose, and protease inhibitors II)

Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA)

Geneticin (Thermo Fisher Scientific)

Reaction buffer (20 mM of Tris-HCl, pH 7.5, 0.25 M sucrose, 5 mM of MgCl₂, 1 mM of MnCl₂, and 10 mM of 2-mercaptoethanol)

Stop buffer (20 mM of Tris-HCl, pH 7.5, 0.25M sucrose, and 5 mM of MgCl₂)

Liquid scintillation counter

HA filters (0.45 μm pore size, 24-mm diameter)

Sampling Manifold (Millipore, MA)

Protocol for the preparation of subcellular fractionation of yeast (Saccharomyces cerevisiae)

a.

Insert cDNA of PAPST into the yeast expression vector YEp352GAP-II with three copies of the HA epitope tag (YPYDVPDYA) at the position corresponding to the C terminus of the PAPST to be expressed (Note 1).

b.

Transform yeast strain W303-1a (MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, and can1-100) using the lithium acetate procedure (9) with a YEp352GAP-II vector carrying the HA-tagged PAPST.

c.

Incubate the transformed yeast cells at 30°C in a synthetic defined medium that lacks uracil to select for transformants; continue the culture until it achieves OD₆₀₀ = 3.0.

d.

Harvest the cells by centrifugation (3,000 ×g, 5 min) and wash with ice-cold 10 mM of NaN₃.

e.

Convert the cells into spheroplasts by incubation at 37°C for 20–30 min in a spheroplast buffer.

f.

Pellet the spheroplasts using a refrigerated centrifuge (3,000 ×g, 5 min) and wash twice with wash buffer to remove traces of zymolyase.

g.

Suspend the cells in ice-cold lysis buffer I and homogenize using a Dounce homogenizer (Note 2).

h.

Centrifuge the lysate at 1,000 ×g for 10 min to remove unlysed cells and cell wall debris.

i.

Centrifuge the supernatant at 10,000 ×g for 15 min at 4°C and collect the pellet as the P10 membrane fraction (ER-rich membrane fraction). Do not discard the supernatant.

j.

Centrifuge the supernatant at 100,000 ×g and collect the pellet as the P100 membrane fraction (Golgi-rich membrane fraction).

k.

Quantify protein yields in the pellets using the conventional method (Note 3).

l.

Use each pellet in a transporter activity assay.

Protocol for the preparation of subcellular fractionation of mammalian cells

a.

Insert cDNA of PAPST into the mammalian expression vector pCXN2 (10) with three copies of the HA epitope tag (YPYDVPDYA) at the position corresponding to the C terminus of the PAPST to be expressed (Note 1).

b.

Transfect 8 µg of pCXN2 carrying with HA-tagged PAPST into cultured mammalian cells using Lipofectamine 2000 reagent in accordance with the manufacturer’s protocol.

c.

Select for transformed cells by adding 600 µg/mL of geneticin to the medium and culture for 1 month after 48 h from transfection.

d.

Harvest the transformed cells, suspend in ice-cold lysis buffer II, and homogenize using 20–40 strokes of a Dounce homogenizer.

e.

Centrifuge the lysate at 1,000 ×g for 10 min to remove intact cells and cell debris.

f.

Centrifuge the supernatant at 7,700 ×g for 10 min at 4°C and collect the pellet as the P10 membrane fraction (ER-rich membrane fraction). Do not discard the supernatant.

g.

Centrifuge the supernatant at 100,000 ×g and collect the pellet as the P100 membrane fraction (Golgi-rich membrane fraction).

h.

Quantify protein yields in the pellets using the conventional method (Note 2).

i.

Use each pellet in a transporter activity assay.

Protocol for PAPST activity assay using each subcellular fractionation

a.

Incubate each pellet described above (100 µg of protein) in 50 µL of reaction buffer that contains 1 µM of radiolabeled PAPS for 5 min at the appropriate temperature for the species of origin of the PAPST (Note 3).

b.

Stop the reaction by adding 1 mL of ice-cold stop buffer (Note 4).

c.

Trap the radioactivity incorporated in the microsomes using 0.45 µm of HA filters, and then, wash the filter with 10 mL of ice-cold stop buffer (Note 4).

d.

Air-dry and measure radioactivity using a liquid scintillation counter. The amount of incorporated radioactivity is calculated as the difference from the background value obtained using the same assay at the appropriate temperature for 0 min for each sample.

An HA tag can be inserted at the position corresponding to the N-terminus of PAPST.

The expression level of HA-tagged PAPST in each membrane fraction is checked by western blotting using an anti-HA mouse mAb (Roche, Basel, Swiss).

Reaction temperature varies with species of origin of the PAPS transporters as follows: C. elegans, 25°C (5); Drosophila, 30°C (1, 3); mouse, 32°C (4); and human, 30°C (1, 2).

Perform each handling step quickly and in a serial order from steps 3b and 3c of Protocol for the transporter activity assay.