Fig. 1. The last 91 residues of Rh50 are sufficient for membrane protein localization on the CV. (A) Schematic representation of the CsA-Rh50 chimeric protein used in this study. The fusion protein is composed of the contact site A external domain (EX), P29F8 transmembrane domain (TM) and a short cytoplasmic domain (CYT) either followed by a stop codon (CsA-Stop) or the 91 C-terminal residues of Rh50 (pFL674). (B) Confocal microscopy study of cells expressing CsA-Stop or CsA-Rh50 (pFL674) constructs. Cells were double-labeled with the anti-CsA antibody (41-71-21) and polyclonal antibodies to either calmodulin or Rh50 (RH153) or the monoclonal antibody to p80 (H161) directly coupled to Alexa Fluor 488. Merged images are shown in the right-hand column. For calmodulin staining, cells were not fixed in paraformaldehyde but directly incubated in –20°C methanol for 10 minutes. CsA-Rh50 colocalized with endogenous Rh50 or with calmodulin, two CV-resident proteins, but was excluded from early and late p80-positive endosomes. Bar, 10 µm.