Fig. 3. CHMP1 physically interacts with human SKD1/VPS4 in vitro and in vivo. (A) CHMP1 and a control GFP protein were radiolabeled by in vitro translation and incubated with purified, immobilized MBP, MBP-SKD1 or MBP-SKD1(EQ). Bound protein was eluted, resolved by SDS-PAGE and visualized by fluorography. The input lane represents one-tenth the amount of radiolabeled protein used in each binding reaction. Note that only CHMP1 efficiently binds both wild-type and mutant (EQ) SKD1. (B) COS7 cells were transiently transfected with plasmids that produce unfused- or SKD1(EQ) -fused FLAG epitope, plus (lanes 1-4) or minus (lanes 5,6) a CHMP1 expression vector. Cell extracts were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma), and bound material was analyzed by SDS-PAGE, followed by western blot with CHMP1 antibody. Note that SKD1(EQ) can immunoprecipitate endogenous CHMP1 (lane 6). (C) Transfected COS7 cells overexpressing GFP-SKD1(EQ) were analyzed by immunocytochemistry for changes in the endogenous CHMP1 distribution. Transfected (arrowhead) and untransfected (arrow) cells are indicated. Cells were visualized by fluorescence microscopy.
