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 cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to 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 with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the 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 range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.
Scotland
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Department of Biochemistry, University of Glasgow, U.K.
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D015500:Chloramphenicol O-Acetyltransferase / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers / Q000737:chemistry; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014162:Transfection
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D015500:Chloramphenicol O-Acetyltransferase; D015105:3',5'-Cyclic-AMP Phosphodiesterases