nup49-313 Shuttling in Yeast
Equipment and reagents
• PEG/TE/LiOAc
• TE/LiOAc
• DMSO
• 2% glucose
• 2% galactose
• Dropout media
• 2% raffinose
• Deionized water
• 25 x 75 x 1 mm frosted microscope slides
• 24 x 40 mm micro cover slips
• Phase contrast and fluorescence microscope
Method
1. Transform a plasmid containing YFG-GFP under the control of the galactose inducible promoter (pGAL1-10)a into a nup49-313 strainb
2. Grow the transformants in 2% glucose containing dropout media to saturation at 25°C
3. Dilute (1:200 dilution) and grow the cells in 2% raffinose containing dropout media at 25°C
4. When the cells reach 1 x 107 cells/ml, add 2% galactose to the cultures and incubate at 25°C for 3 hours.
5. Wash the cells once with dH2O and resuspend the cells in dropout media containing 2% glucose.
6. Incubate the cells at 25°C for 2 hours.
7. Split each culture and incubate one half of the culture at 25°C and the other half of the culture at 37°C for 5 hours.
8. Remove 1 ml of cells from each culture, wash once with dH2O, and resuspend in 25-100 ml of dH2O
9. Place 3 ml of washed cells onto a glass slide and analyze nuclear export by fluorescence microscopy.
Alternatively, cells expressing YFG-GFP under the control of its endogenous promoter (pYFG) can be used in the assay. Steps 3-6 should be modified to include shifting the cells for 2-3 hours until they are in mid log phase followed by incubation of the cells with 100 mM cyclohexamide before splitting and shifting the cells.
This assay can be coupled with other mutants to examine whether other proteins affect nuclear export of YFG-GFP
[Silver, 199_]
