Transport of UDP-[3H]-Glc to Golgi-Vesicles

TRANSPORT OF UDP-[3H]-GLC TO GOLGI-VESICLES

Last Update: December 2006

Based on Munoz P, Norambuena L, Orellana A (1996) Evidence for a UDP-glucose transporter in Golgi apparatus-derived vesicles from pea and its possible role in polysaccharide biosynthesis. Plant Physiol 112: 1585-1594

PREPARE SOLUTIONS

1. 2X STM buffer (500 mL):

Mix 80 g of sucrose (0.5M), 1 mL of 1M MgCl₂ (2mM), 10 mL of 1M Tris-HCl, pH 7.5 (20mM), and dH₂O to 500 mL. Aliquot and store at -20^oC

PROCEDURE

1. Set up a multi-channel filter system capable of holding 25mm Glass-fiber filter disks (0.7mm, Sigma-Aldrich, F7786, 100% borosilicate glass)

2. Hydrate filters with 1X STM

3. Set up trasport reactions: 100 mg of Golgi vesicle protein, 10-100,000 dpms of UDP-[³H]-Glc (or GST-AtRGP1-[³H]-Glc), UDP-Glc to a final concentration of 1mM, all in 1X STM buffer

4. Incubate at 25^oC (or 0^oC) for differents times and stop reaction with:

4.1 Filtering through 0.7 mm filters (to measure transport of label to Golgi vesicles)

4.2 Add 9 volumes of 80% EtOH, incubate on ice for 30 minutes, and filter through a 1.5mm filter, followed by two washes with 70% EtOH (to measure polysaccharide synthesis)

4.3 Add TCA to a final concetration of 10%, incubate on ice for 30 minutes, and filter through a 1.5mm filter, followed by two washes with 10% TCA (to measure glycoprotein synthesis)

5. Let filters dry on top of aluminum foil and then place on a scintillation counting vial with 2 mL of scintillation liquid

6. Count on a scintillation counter