Assay of Nucleotide Sugar Transport Activity (Golgi-ER transporter)

Authors：

Category

Nucleotide sugar transporters

Protocol Name

Authors

Nakayama, Ken-ichi
Biomass Treatment Group, Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST)

Shimma, Yoh-ichi
Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST)

Oka, Takuji
Faculty of Biotechnology and Life Science, Department of Applied Microbial Technology, Sojo University

Nishihara, Shoko *
Laboratory of Cell Biology, Department of Bioinformatics, Graduate School of Engineering, Soka University

*To whom correspondence should be addressed.

KeyWords

Nucleotide sugar transporter activity Nucleotide sugar Sugar nucleotide

Reagents

●	Nucleotide sugar
●	Radio labeled nucleotide sugar (ex. UDP-[³H]GlcNAc)
●	Zymolyase (Zymolyase-100T; Seikagaku Corp., Tokyo, Japan)
●	Protease inhibitors (Complete protease inhibitor mixture tablets; Roche Applied Science, Penzberg, Germany)
●	Synthetic defined medium
●	Spheroplast buffer (1.4 M sorbitol, 50 mM potassium phosphate pH 7.5, 10 mM NaN₃, 40 mM 2-mercaptoethanol, Zymolyase 100 T 1mg/g wet cells)
●	Wash buffer (1.4 M sorbitol)
●	Lysis buffer (0.6 M sorbitol, 10 mM triethanolamine pH 7.2, protease inhibitors)
●	Reaction mixture (20 mM Tris-HCl pH 7.5, 0.25 M sucrose, 5 mM MgCl₂, 1mM MnCl₂, 10 mM 2-mercaptoethanol)
●	Stop buffer (20 mM Tris-HCl pH 7.5, 0.25 M sucrose, 150 mM KCl, 1 mM MgCl₂)

Instruments

●	Liquid scintillation counter
●	HA filters (0.45 μm pore size, 24 mm diameter)
●	Sampling Manifold (1225, Merck Millipore, Billerica, MA)

Methods

Preparation of ER or Golgi-rich membrane fraction

1)	Insert cDNA of nucleotide sugar transporter into the yeast expression vector YEp352GAP-II.	Comment 0

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.	Comment 0

3)	Incubate the transformed yeast cells at 30˚C in a synthetic defined medium that lacks uracil in order to select for transformants; continue culture until an OD₆₀₀ of approximately 3.0 is achieved.	Comment 0

4)	Collect yeast cells by centrifugation (3,000 × g, 5 min).	Comment 0

5)	Wash with ice-cold 10 mM NaN₃.	Comment 0

6)	Suspend in 30 mL of spheroplast buffer.	Comment 0

7)	Incubate at 30˚C for 35 min.	Comment 0

8)	Centrifuge (3,000 × g, 5 min) and collect the pellet.	Comment 0

9)	Wash 3 times with wash buffer.	Comment 0

10)	Add 20 mL of lysis buffer.	Comment 0

11)	Homogenize using Dounce homogenizer.	Comment 1

12)	Centrifuge at 1,000 × g for 10 min and collect supernatant.	Comment 0

13)	Centrifuge the supernatant at 10,000 × g for 15 min and collect pellet as the P10 membrane fraction (ER-rich membrane fraction). Do not discard the supernatant.	Comment 0

14)	Centrifuge the above supernatant at 100,000 × g for 1 h and collect the pellet as the P100 membrane fraction (Golgi-rich membrane fraction).	Comment 0

15)

Quantify protein contents of the fractions by the conventional method.

Comment 0

Assay of nucleotide sugar transporter activity

1)	Suspend 240 μg proteins of P10 or P100 membrane fraction in 100 μL of reaction mixture with radio labeled nucleotide sugar.	Comment 0

2)	Incubate at 30˚C for 5 min.	Comment 0

3)	Add 1 mL of ice-cold stop buffer.	Comment 1

4)	Filter through 0.45 μm HA filters.	Comment 1

5)	Wash with 10 mL of stop buffer.	Comment 1

6)	Air-dry and place in vial with 4 mL of scintillation mixture.	Comment 0

Count using liquid scintillation counter.

Comment 0

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Date of registration：2016-07-14 11:49:59