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A mobile loop near the active site acts as a switch between the dual activities of a viral protease/deubiquitinase

Fig 4

Impact of WT or mutant 98K proteins on ubiquitylation of 66K protein.

Arabidopsis protoplasts were transfected with pΩ-66K, pΩ-myc2-Ub, alone or together with pΩ-98K, pΩ-98K-C783S, pΩ-98K-P866G/P867G, pΩ-98K-G865A, pΩ-98K-D843A, pΩ-98K-I847A or pΩ-98K-I847D as indicated. Cells were collected 48 hpt, and samples were immunoprecipitated under denaturing conditions with anti-66K antibody. Samples were then normalized according to the amount of 66K and subjected to immunoblotting with anti-myc antibody (IP 66K / WB myc). The positions of molecular mass markers are indicated on the left, and that of viral proteins on the right. Arrowhead: Position of 66K. The amount of 66K present in the immunoprecipitates was determined by immunoblotting with anti-66K antibody (IP 66K / WB 66K), and the amount of 98K derivatives present in the cell lysates was determined by immunoblotting with anti-98K antibody prior to immunoprecipitation (lysate / WB 98K). The shorter 85K protein corresponds to a nonspecific degradation product of 98K occurring during sample extraction and protein analysis [31]. Ponceau staining of the membrane (lysate staining) indicates protein loading in the whole cell lysate samples. Each mutant was analyzed in two independent experiments.

Fig 4