Cloning and characterization of beta-COP from Dictyostelium discoideum

We have isolated a cDNA coding for beta-COP from Dictyostelium discoideum b y polymerase chain reaction using degenerate primers derived front rat beta -COP. The complete cDNA clone has a size of 2.8kb and codes for a protein w ith a calculated molecular mass of 102 kDa. Dictyostelium beta-COP exhibits highest homology to mammalian beta-COP, but it is considerably smaller due to a shortened variable region that is thought to form a linker between th e highly conserved N- and C-terminal domains, Dictyostelium beta-COP is enc oded by a single gene, which is transcribed at moderate levels into two RNA s that are present throughout development. To localize the protein, full-le ngth beta-COP was fused to GFP and expressed in Dictyostelium cells, The fu sion protein was detected on vesicles distributed all over the cells and wa s strongly enriched in the perinuclear region Based on coimmunofluorescence studies with antibodies directed against the Golgi marker comitin, this co mpartment was identified as the Golgi apparatus, beta-COP distribution in D ictyostelium was not brefeldin A sensitive being most likely due to the pre sence of a brefeldin A resistance gene. However, upon DMSO treatment we obs erved a reversible disassembly of the Golgi apparatus. In mammalian cells D MSO treatment had a similar effect on beta-COP distribution.