CHIMERIC CONSTRUCTS SHOW THAT THE UNIQUE N-TERMINAL DOMAIN OF THE CYCLIC-AMP PHOSPHODIESTERASE RD1 (RNPDE4A1A RPDE-IVA1) CAN CONFER MEMBRANE ASSOCIATION UPON THE NORMALLY CYTOSOLIC PROTEIN CHLORAMPHENICOL ACETYLTRANSFERASE

A novel plasmid was generated which allowed the expression of the cyto solic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS -7 cells. Upon transfection, the majority of the novel CAT activity wa s found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific r at 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused t o CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated w ith the membrane fraction. In contrast, a chimera formed from 26-100RD 1-CAT showed an identical expression pattern to native CAT, with the m ajor fraction of CAT activity occurring in the cytosol fraction. Membr ane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was no t released by either high-salt or washing treatments but was solubiliz ed in a dose-dependent fashion by the non-ionic detergent Triton X-100 . Subcellular fractionation of COS-7 cells showed that, as with RD1, t he membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5'-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a rang e of N-terminal truncated species due to initiation at different methi onine residues. Incubation of the mature protein products formed in th is system with a COS cell membrane fraction showed that only those chi meric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD 1 contains structural information that can confer membrane association upon an essentially soluble protein.