The nucleotide sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. Sulfation of proteins and glycans is performed in the Golgi lumen by various sulfotransferases. In similar fashion to nucleotide sugars, PAPS is synthesized in the cytosol and translocated from the cytosol into the Golgi lumen via a PAPS transporter. (Kamiyama et al. 2003; Goda et al. 2006; Kamiyama et al. 2006; Sasaki et al., 2009; Dejima et al., 2010). Therefore, PAPS transporters belong to the nucleotide sugar transporter family and PAPS transporter activity can be measured by similar methods to those for nucleotide sugar transporters.
There are two types of assay for nucleotide sugar transporter activity: the ‘heterologous expression system’(Kamiyama et al. 2003) and the ‘proteoliposome system’*1. The former makes use of expression of human nucleotide sugar transporter in a yeast expression system, and is based on the fact that the yeast microsome normally shows only low endogenous nucleotide sugar transporter activity, except for GDP-Man transporter activity. In this chapter, we will describe two protocols for determining PAPS transporter activity: 1) the yeast expression system; and 2) the mammalian expression system.
*1. Refer to “Caffaro CE and Hirschberg CB (2006) Nucleotide sugar transporters of the Golgi apparatus: from basic science to diseases. Acc Chem Res. 39: 805-812". [PMID : 17115720] |
| Category | Nucleotide sugar transporters |
| Protocol Name | Assay of PAPS Transport Activity |
Authors
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Nishihara, Shoko
Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University
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| KeyWords |
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Reagents
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Radio labeled PAPS ([35S]PAPS:1.66Ci/mmol; PerkinElmer, Waltham, MA) |
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Zymolyase (Zymolyase-100T; Seikagaku Biobusiness Corp., Tokyo, Japan) |
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Protease inhibitors I (5 μg/mL pepstatin A, and 1 mM phenylmethylsulfonyl fluoride) |
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Protease inhibitors II (1 mM phenylmethylsulfonyl fluoride, 1 μg/mL leupeptin, 1 μg/mL aprotinin, 1 μg/mL pepstatin A) |
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Spheroplast buffer (1.4 M sorbitol, 50 mM potassium phosphate pH7.5, 10 mM NaN3, 40 mM 2-mercaptoethanol, Zymolyase 100 T 1 mg/g wet cells) |
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Wash buffer (1.0 M sorbitol) |
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Lysis buffer I (0.8 M sorbitol, 10 mM triethanolamine pH7.2, protease inhibitors I) |
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Lysis buffer II (10 mM HEPES-Tris pH7.4, 0.25 M sucrose, protease inhibitors II) |
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Lipofectamine 2000 (Invitrogen/Lifetechnologies, Carlsbad, CA) |
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Geneticin (Invitrogen/Life Technologies) |
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Reaction buffer (20 mM Tris-HCl pH7.5, 0.25 M sucrose, 5 mM MgCl2, 1 mM MnCl2, 10 mM 2-mercaptoethanol) |
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Stop buffer (20 mM Tris-HCl pH7.5, 0.25 M sucrose, 5 mM MgCl2) |
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Instruments
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Liquid scintillation counter |
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HA filters (0.45 μm pore size, 24 mm diameter) |
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Sampling Manifold (1225, Merck Millipore, Billerica, MA) |
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| Methods |
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1. |
Protocol for subcellular fractionation of yeast (Saccharomyces cerevisiae)
(Kamiyama et al. 2003; Goda et al. 2006; Kamiyama et al. 2006; Sasaki et al. 2009) |
| 1) |
Insert cDNA of PAPS transporter into the yeast expression vector YEp352GAP-II with three copies of HA epitope tags (YPYDVPDYA) at the position corresponding to the C terminus of the PAPS transporter to be expressed. |
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| 2) |
Transform yeast strain W303-1a (MATa, ade2-1, ura3-1, his3-11,15, trp1-1, leu2-3,112, and can1-100) by the lithium acetate procedure with a YEp352GAP-II vector carrying the HA-tagged PAPS transporter. |
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Incubate the transformed yeast cells at 30˚C in synthetic defined medium that lacks uracil in order to select for transformants; continue the culture until it achieves OD600 = 3.0. |
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Harvest the cells by centrifugation (3,000 x g, 5 min) and wash with ice-cold 10 mM NaN3. |
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Convert the cells into spheroplasts by incubation at 37˚C for 20-30 min in spheroplast buffer. |
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Pellet the spheroplasts using a refrigerated centrifuge (3,000 x g, 5 min) and wash twice with wash buffer to remove traces of zymolyase. |
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Suspend the cells in ice-cold lysis buffer I and homogenize using a Dounce homogenizer. |
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Centrifuge the lysate at 1,000 x g for 10 min to remove unlysed cells and cell wall debris. |
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Centrifuge the supernatant at 10,000 x 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. |
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Centrifuge the supernatant at 100,000 x g and collect the pellet as the P100 membrane fraction (Golgi-rich membrane fraction). |
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Quantify protein yields in the pellets by the conventional method. |
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Use each pellet in a transporter activity assay. |
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2. |
Protocol for subcellular fractionation of mammalian cells
(Kamiyama et al. 2006) |
| 1) |
Insert cDNA of PAPS transporter into the mammalian expression vector pCXN2* with three copies of HA epitope tags (YPYDVPDYA) at the position corresponding to the C terminus of the PAPS transporter to be expressed**. |
Comment 1
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Transfect 8 μg of pCXN2 carrying with HA-tagged PAPS transporter into cultured mammalian cells using Lipofectamine 2000 reagent in accordance with the manufacturer’s protocol. |
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Select for transformed cells by adding 600 μg/mL of geneticin to the medium and culture for 1 month after 48 h from transfection. |
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Harvest the transformed cells, suspend in ice-cold lysis buffer II and homogenize using a Dounce homogenizer. |
Comment 1
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Centrifuge the lysate at 1,000 x g for 10 min to remove unlysed cells and cell wall debris. |
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| 6) |
Centrifuge the supernatant at 7,700 x 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. |
Comment 0
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| 7) |
Centrifuge the supernatant at 100,000 x g and collect the pellet as the P100 membrane fraction (Golgi-rich membrane fraction). |
Comment 0
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| 8) |
Quantify protein yields in the pellets by the conventional method. |
Comment 0
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| 9) |
Use each pellet in a transporter activity assay. |
Comment 1
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3. |
Protocol for transporter activity assay
(Kamiyama et al. 2003; Goda et al. 2006; Kamiyama et al. 2006; Sasaki et al. 2009) |
| 1) |
Incubate each of the pellets described above (100 μg protein) in 50 μL of reaction buffer that contains 1 μM radiolabeled PAPS for 5 min at the appropriate temperature for the species of origin of the PAPS transporter. |
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Stop the reaction by adding 1 mL of ice-cold stop buffer. |
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Trap the radioactivity incorporated in the microsomes using 0.45 μm HA filters and then wash the filter with 10 mL of ice-cold stop buffer. |
Comment 1
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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. |
Comment 0
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| Copyrights |
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This work is released underCreative Commons licenses
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| Date of registration：2013-12-25 10:21:05 |
- Kamiyama, S., Suda, T., Ueda, R., Suzuki, M., Okubo, R., Kikuchi, N., Chiba, Y., Goto, S., Toyoda, H., Saigo, K., Watanabe, M., Narimatsu, H., Jigami, Y., Nishihara, S. (2003) Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter. J Biol Chem 278: 25958-25963. [PMID : 12716889]
- Kamiyama, S., Sasaki, N., Goda, E., Ui-Tei, K., Saigo, K., Narimatsu, H., Jigami, Y., Kannagi, R., Irimura, T., Nishihara, S. (2006) Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2. J Biol Chem 281: 10945-10953. [PMID : 16492677]
- Goda, E., Kamiyama, S., Uno, T., Yoshida, H., Ueyama, M., Kinoshita-Toyoda, A., Toyoda, H., Ueda, R., Nishihara, S. (2006) Identification and characterization of a novel Drosophila 3'-phosphoadenosine 5'-phosphosulfate transporter. J Biol Chem 281: 28508-28517. [PMID : 16873373]
- Sasaki, N., Hirano, T., Ichimiya, T., Wakao, M., Hirano, K., Kinoshita-Toyoda, A., Toyoda, H., Suda, Y., Nishihara, S. (2009) The 3'-phosphoadenosine 5'-phosphosulfate transporters, PAPST1 and 2, contribute to the maintenance and differentiation of mouse embryonic stem cells. PLoS One, 4: e8262. [PMID : 20011239]
- Dejima, K., Murata, D., Mizuguchi, S., Nomura, KH., Izumikawa, T., Kitagawa, H., Gengyo-Ando, K., Yoshina, S., Ichimiya, T., Nishihara, S., Mitani, S., Nomura, K.: (2010) Two Golgi-resident 3' -phosphoadenosine 5' -phosphosulfate transporters play distinct roles in heparan sulfate modifications and embryonic and larval development in Caenorhabditis elegans. J Biol Chem, 285: 24717-24728. [PMID : 20529843]
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