FIGURE 4.

GRAIL ubiquitinates intracellular lysine residues only present in the tetraspanin amino terminus. A, ClustalW alignment of the amino terminus of human tetraspanins and diagram of CD81 and CD151 lysine point mutants constructed. ^* designates conserved lysine residue. B, 293T cells were transfected with either V5-tagged GRAIL or vector alone, 3×HA-tagged CD81 or CD151, and 3×FLAG-tagged ubiquitin (Ub). Cell lysates were denatured in SDS, immunoprecipitated (IP) with anti-HA beads, and immunoblotted (IB) with anti-HA and anti-FLAG. C, 293T cells were transfected with 0.5 μg of 3×HA-tagged CD81 K8R,K11R and V5-tagged GRAIL at 0, 0.4, 0.8, 1.2, 1.6, and 2 μg (empty V5 vector was added for a total of 2 μg of total V5 plasmid for each transfection). Cell lysates were separated by SDS-PAGE and blotted with the indicated antibodies.