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Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-.
Introduction
Sulfations of proteins and glycans are performed in the Golgi lumen by various kinds of sulfotransferases. The nucleotide sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), is a universal sulfuryl donor for sulfation. In similar fashion to nucleotide sugars, PAPS is synthesized in the cytosol and subsequently translocated into the Golgi lumen via a PAPS transporter (PAPST) (1–6). Currently, two types of PAPSTs have been cloned, and their activities have been identified (1, 2). PAPSTs are multiple membrane-spanning proteins that transport PAPS by a coupled antiport of adenosine 3', 5'-diphosphate (PAP) (Figure 1). Therefore, PAPSTs determine the sulfation status, as they supply the sulfate groups necessary as a substrate for sulfotransferases.
PAPSTs are members of the solute carrier 35 (SLC35) transporter family, which also includes nucleotide sugar transporters (Figure 2). PAPST activity can be measured by the methods similar to that of nucleotide sugar transporter.
There are two types of assays for nucleotide sugar transporter activity: the “heterologous expression system” (1, 7) and the “proteoliposome system” (8). The former method uses the expression of nucleotide sugar transporter in a yeast expression system because the yeast microsome normally shows only low endogenous nucleotide sugar transporter activity except for GDP-Man transporter activity.
Protocol
In this chapter, the two types of protocols will be described for the determination of PAPST activity: 1) by yeast expression system and 2) by mammalian expression system.
Materials
- 1.
Radio labeled PAPS ([35S] PAPS:1.66Ci/mmol)
- 2.
Zymolyase (Zymolyase-100T; Seikagaku Biobusiness, Tokyo, Japan)
- 3.
Protease inhibitors I (5 µg/mL of pepstatin A and 1 mM of phenylmethylsulfonyl fluoride)
- 4.
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)
- 5.
- 6.
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)
- 7.
Wash buffer (1.0 M sorbitol)
- 8.
Lysis buffer I (0.8 M sorbitol, 10 mM of triethanolamine, pH7.2, and protease inhibitors I)
- 9.
Lysis buffer II (10 mM of HEPES-Tris, pH 7.4, 0.25 M sucrose, and protease inhibitors II)
- 10.
Lipofectamine 2000 (Thermo Fisher Scientific, MA, USA)
- 11.
Geneticin (Thermo Fisher Scientific)
- 12.
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)
- 13.
Stop buffer (20 mM of Tris-HCl, pH 7.5, 0.25M sucrose, and 5 mM of MgCl2)
Instruments
- 1.
Liquid scintillation counter
- 2.
HA filters (0.45 μm pore size, 24-mm diameter)
- 3.
Sampling Manifold (Millipore, MA)
Methods
- 1.
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.
- 2.
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.
- 3.
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.
Notes
- 1.
An HA tag can be inserted at the position corresponding to the N-terminus of PAPST.
- 2.
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).
- 3.
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).
- 4.
Perform each handling step quickly and in a serial order from steps 3b and 3c of Protocol for the transporter activity assay.
References
- 1.
- 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. Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter. J Biol Chem. 2003 Jul 11;278(28):25958–63. [PubMed: 12716889] [CrossRef]
- 2.
- Kamiyama S, Sasaki N, Goda E, Ui-Tei K, Saigo K, Narimatsu H, Jigami Y, Kannagi R, Irimura T, Nishihara S. Molecular cloning and characterization of a novel 3'-phosphoadenosine 5'-phosphosulfate transporter, PAPST2. J Biol Chem. 2006 Apr 21;281(16):10945–53. [PubMed: 16492677] [CrossRef]
- 3.
- Goda E, Kamiyama S, Uno T, Yoshida H, Ueyama M, Kinoshita-Toyoda A, Toyoda H, Ueda R, Nishihara S. Identification and characterization of a novel Drosophila 3'-phosphoadenosine 5'-phosphosulfate transporter. J Biol Chem. 2006 Sep 29;281(39):28508–17. [PubMed: 16873373] [CrossRef]
- 4.
- Sasaki N, Hirano T, Ichimiya T, Wakao M, Hirano K, Kinoshita-Toyoda A, Toyoda H, Suda Y, Nishihara S. The 3'-phosphoadenosine 5'-phosphosulfate transporters, PAPST1 and 2, contribute to the maintenance and differentiation of mouse embryonic stem cells. PLoS One. 2009 Dec 11;4(12):e8262. [PMC free article: PMC2788424] [PubMed: 20011239] [CrossRef]
- 5.
- 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. 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. 2010 Aug 6;285(32):24717–28. [PMC free article: PMC2915708] [PubMed: 20529843] [CrossRef]
- 6.
- Kamiyama S, Ichimiya T, Ikehara Y, Takase T, Fujimoto I, Suda T, Nakamori S, Nakamura M, Nakayama F, Irimura T, Nakanishi H, Watanabe M, Narimatsu H, Nishihara S. Expression and the role of 3'-phosphoadenosine 5'-phosphosulfate transporters in human colorectal carcinoma. Glycobiology. 2011 Feb;21(2):235–46. [PubMed: 20978009] [CrossRef]
- 7.
- Suda T, Kamiyama S, Suzuki M, Kikuchi N, Nakayama K, Narimatsu H, Jigami Y, Aoki T, Nishihara S. Molecular cloning and characterization of a human multisubstrate specific nucleotide-sugar transporter homologous to Drosophila fringe connection. J Biol Chem. 2004 Jun 18;279(25):26469–74. [PubMed: 15082721] [CrossRef]
- 8.
- Caffaro CE, Hirschberg CB. Nucleotide sugar transporters of the Golgi apparatus: from basic science to diseases. Acc Chem Res. 2006 Nov;39(11):805–12. [PubMed: 17115720] [CrossRef]
- 9.
- Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–8. [PMC free article: PMC217353] [PubMed: 6336730] [CrossRef]
- 10.
- Niwa H, Yamamura K, Miyazaki J. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991 Dec 15;108(2):193–9. [PubMed: 1660837] [CrossRef]
Footnotes
The authors declare no competing or financial interests.
- Analysis of 3'-Phosphoadenosine 5'-Phosphosulfate Transporters: Transporter Activity Assay, Real-Time Reverse Transcription Polymerase Chain Reaction, and Immunohistochemistry.[Methods Mol Biol. 2022]Analysis of 3'-Phosphoadenosine 5'-Phosphosulfate Transporters: Transporter Activity Assay, Real-Time Reverse Transcription Polymerase Chain Reaction, and Immunohistochemistry.Egawa H, Nishihara S. Methods Mol Biol. 2022; 2303:675-685.
- Review Sulfation and sulfotransferases 5: the importance of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the regulation of sulfation.[FASEB J. 1997]Review Sulfation and sulfotransferases 5: the importance of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) in the regulation of sulfation.Klaassen CD, Boles JW. FASEB J. 1997 May; 11(6):404-18.
- Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter.[J Biol Chem. 2003]Molecular cloning and identification of 3'-phosphoadenosine 5'-phosphosulfate transporter.Kamiyama S, Suda T, Ueda R, Suzuki M, Okubo R, Kikuchi N, Chiba Y, Goto S, Toyoda H, Saigo K, et al. J Biol Chem. 2003 Jul 11; 278(28):25958-63. Epub 2003 Apr 25.
- Depletion of hepatic 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and sulfate in rats by xenobiotics that are sulfated.[J Pharmacol Exp Ther. 1995]Depletion of hepatic 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and sulfate in rats by xenobiotics that are sulfated.Kim HJ, Cho JH, Klaassen CD. J Pharmacol Exp Ther. 1995 Nov; 275(2):654-8.
- Review [Production and application of 3-phosphoadenosine-5- phosphosulfate].[Sheng Wu Gong Cheng Xue Bao. 2...]Review [Production and application of 3-phosphoadenosine-5- phosphosulfate].Zhou Z, Du G, Kang Z. Sheng Wu Gong Cheng Xue Bao. 2019 Jul 25; 35(7):1222-1233.
- Assay of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transport activity - Glyco...Assay of 3'-phosphoadenosine 5'-phosphosulfate (PAPS) transport activity - Glycoscience Protocols (GlycoPODv2)
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