Glucosylation in Cell Lysates

Before cell lysis, the cell monolayer is rinsed with ice cold phosphate-buffered saline (PBS) and the cells are then mechanically removed in the presence of lysis buffer (2 mM MgCb, 0.1 mM phenylmethylsulfo-nyl fluoride, 20jig/ml leupeptin, 80n.g/ml benzamidine in 50 mM HEPES, pH 7.4) followed by sonication five times on ice. After centrifugation for 10 min at 2000 g to remove the nuclei and cell debris, the supernatant is used for the glucosylation reaction. 

40 xl of cell lysate plus 10n.M of UDP-[ C]glucose (300mCi/ mmol) and 20 [iM unlabeled UDP-glucose, is incubated with 1 (xg/ml of toxin B (total volume 50 [xl) for 45 min at 37°C. If UDP-[ Cjglucose is purchased in ethanolic solution, a sample is freeze dried and dissolved in 50 mM HEPES, pH 7.4 to obtain a concentration of 100 fxM. 

The glucosylation reaction is terminated by addition of 10 [xl Laemmli sample buffer followed by incubation for 5 min at 95°C. Alternatively, the reaction is stopped by addition of 1 ml of trichloroacetic acid (20%, w/v). 

Toxin A glucosylates the identical substrate proteins (Rho, Rac, Cdc42) that are glucosylated by toxin B. Thus, the glucosylation reaction can also be performed with toxin A. However, the toxin A concentration has to be 10(ig/ml (instead of 1 (ig/ml for toxin B). 

Recombinant Rho proteins are glucosylated in the presence of unlabeled UDP-glucose. The reaction is terminated by passing the reaction mixture through a membrane with an exclusion molecular weight of 100 kDa to remove toxin B (270 kDa). As a control, the GTPase is incubated with toxin B but in absence of the cosubstrate UDP-glucose. 

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