HUMAN N-MYRISTOYLTRANSFERASE AMINO-TERMINAL DOMAIN INVOLVED IN TARGETING THE ENZYME TO THE RIBOSOMAL SUBCELLULAR FRACTION

N-Myristoyltransferase (NMT) catalyzes the cotranslational acylation w ith myristic acid of the NH2-terminal glycines of a number of cellular and viral proteins. Most of the in vitro NMT activity (60-85%) in iso osmotic cell homogenates of human lymphoblastic leukemia (i.e. CEM and MOLT-4) and cervical carcinoma (i.e. HeLa) cells was shown to be asso ciated with the ribosomal subcellular fractions by differential centri fugation. Also found in the ribosomal fractions was a approximate to 6 0-kDa protein that was specifically immunoblotted with an anti-human N MT (hNMT) peptide antibody. This approximate to 60-kDa protein was sta ble in the presence of proteolytic enzyme inhibitors but was gradually converted into a approximate to 46-kDa species when stored in the abs ence of protease inhibitors. Sucrose density gradient centrifugation o f the ribosomal fraction resulted in the hNMT activity sedimenting exa ctly coincident with the 260 nm absorption profile and exhibiting A(26 0)/A(260) absorption ratios >1.8, indicating an association of NMT wit h putative ribosomal particle (s)/subunit(s), The subcellular targetin g of hNMT was also examined by immunoblotting subcellular fractions fr om HeLa cells transfected with plasmids containing FLAG epitope-tagged hNMT inserts corresponding either to the originally assigned hNMT gen e or to an alternative open reading frame initiated from an in-frame s tart site upstream from the assumed hNMT start site. Anti-FLAG immunob lotting of cells transfected with a plasmid containing the larger inse rt revealed FLAG-NMT primarily in the ribosomal fraction with an appar ent molecular mass similar to the approximate to 60-kDa native hNMT. I n contrast, immunoblotting of cells transfected with a plasmid contain ing the smaller insert identified a approximate to 50-kDa FLAG-NMT pre dominantly in the cytosolic fraction. An analysis of mixtures of CEM r ibosomes and serial dilutions of purified recombinant FLAG-NMTs demons trated that the approximate to 60-kDa FLAG-NMT binds ribosomes with hi gher affinity than the approximate to 50-kDa FLAG-NMT. These in vivo a nd-in vitro subcellular targeting and recombinant expression experimen ts identify a native hNMT that is 10-12 kDa larger than the enzyme pre dicted by the originally assigned hNMT gene and which is apparently tr anslated from an alternative up stream start site. The data also indic ate that although the unique NH2-terminal residues encoded by this lar ger open reading frame are not required for in vitro catalytic activit y, they do provide signal(s) involved in targeting hNMT to the ribosom al subcellular fraction where cotranslational N-myristoylation occurs.