Cloning and characterization of a Dictyostelium discoideum cDNA encoding a protein related to the medium chain subunit of clathrin-associated adaptor complexes.

We describe the cloning and characterization of a cDNA, DdApm1, encoding a putative medium chain subunit of a clathrin-associated protein (adaptor or assembly protein [AP]) complex in Dictyostelium discoideum. The DdApm1 clone is predicted to encode a polypeptide of 439 amino acids (aa) with a molecular mass of 49.9 kDa. The predicted translation product (DdApm1p) shares at least 51.7% identity and 76.3% similarity with the medium chain subunits of plasma membrane (mb)-associated clathrin AP complexes from rat and Caenorhabditis elegans. The deduced aa sequence also demonstrates significant but lesser homology to a number of medium chain subunits of Golgi-associated clathrin AP complexes. Since DdApm1p demonstrates significantly greater homology to plasma mb-associated clathrin AP complex medium chains than to their Golgi-associated counterparts, we suggest that DdApm1p may be a medium chain subunit of an AP complex involved in clathrin function at the plasma mb of D. discoideum. Southern blot analysis indicated that DdApm1 gene defines a single copy gene in the D. discoideum genome. Northern blot analysis of RNA purified at different times during growth and development demonstrated that the DdApm1 gene is expressed at relatively constant levels throughout the life cycle of the organism. DdApm1 is the first reported full-length cDNA encoding a subunit of an AP complex in D. discoideum, and thus provides the first evidence for the existence of AP complexes in this organism.