Figure 3

CpnA binds membranes in a calcium-dependent manner. A) A purified bacterially expressed fragment of CpnA containing only the first C2 domain (CpnA-C2A, 8 μg) was incubated with brain lipid liposomes in the presence of 2 mM calcium (Ca + lipids) or 2 mM EGTA (EGTA + lipids). The liposomes were centrifuged and liposomal pellets were analyzed on an 8% polyacrylamide-SDS gel stained with Coomassie Blue. The first two lanes are controls: 8 μg of CpnA-C2A was loaded on the gel (Control) in the first lane and protein pelleted in the presence of calcium, but absence of lipids (Ca) is in the second lane. B) Wildtype cells were disrupted with passage through a French Press and all membranes were pelleted in the presence of calcium or EGTA. Pellets (P) and supernatants (S) were analyzed by Western blot using CpnA antisera. C) Same procedure as in B) with cells expressing GFP-CpnA. The top arrow points to the GFP-CpnA band, while the bottom arrow points to the CpnA band. D) Same samples as in C) using a monoclonal antibody to GFP.