Fig. 1.

Alanine scanning mutagenesis of laforin and its effect on interaction with malin. A) pEG202-laforin plasmids containing the described mutations were introduced into CTY10-5d yeast strain in combination with plasmid pACT2-malin. Transformants were grown in 4% glucose containing medium until they reach exponential phase. Then, an aliquot of the cultures was shifted to low glucose conditions (0.05% glucose) for 3 hours. Cells from both growth conditions were used to determine the β-galactosidase activity, as an indication of two-hybrid interaction (see Experimental section). Results are the mean values of four to six different transformants, with a standard deviation of less than 15% in each case. B) The two-hybrid interaction between the indicated plasmids was measured as in (A); bars indicate standard deviation. Yeast crude extract (15 μg) from the different transformants were analyzed by western blotting using anti-LexA and anti-HA antibodies. Molecular size standards are in the left. C) The twohybrid interaction between the corresponding protein fusions was measured as above in cells growing in 4% glucose; bars indicate standard deviation. Yeast crude extract (15 μg) from the different transformants were analyzed by western blotting using anti-LexA and anti-HA antibodies.