Fig. 3. A central region of Cos2 contains a membrane binding domain.

A, S2 cells were transfected with expression vectors containing HA-tagged Cos2 (residues 1–1201), Cos2ΔC, Cos2ΔN, or Cos2M. Two days post-transfection, cells were lysed in hypotonic lysis buffer. Total lysate (Tot), postnuclear cytosol (Cyto), and total membrane (Memb) fractions were normalized to volume, separated by SDS-PAGE, and immunoblotted with α-HA antibody (top panel). An immunoblot of endogenous Cos2 is also shown (bottom panel) to demonstrate that endogenous Cos2 is not displaced by overexpressed Cos2. B, S2 cells transfected with eGFP-Cos2ΔN were lysed, and total membranes were separated by equilibrium density centrifugation. eGFP-Cos2ΔN fractionates in a manner similar to endogenous Cos2. C, electron microscopy of the vesicular membrane fraction (fraction 6) compared with the pellet (fraction 10) of the sucrose gradient in B.