Human N-Myristoyltransferase Amino-terminal Domain Involved in Targeting the Enzyme to the Ribosomal Subcellular Fraction*

N-Myristoyltransferase (NMT) catalyzes the cotranslational acylation with 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 isoosmotic cell homogenates of human lymphoblastic leukemia (i.e. CEM and MOLT-4) and cervical carcinoma (i.e. HeLa) cells was shown to be associated with the ribosomal subcellular fractions by differential centrifugation. Also found in the ribosomal fractions was a ≈60-kDa protein that was specifically immunoblotted with an anti-human NMT (hNMT) peptide antibody. This ≈60-kDa protein was stable in the presence of proteolytic enzyme inhibitors but was gradually converted into a ≈46-kDa species when stored in the absence of protease inhibitors. Sucrose density gradient centrifugation of the ribosomal fraction resulted in the hNMT activity sedimenting exactly coincident with the 260 nm absorption profile and exhibitingA 260/A 280 absorption ratios >1.8, indicating an association of NMT with putative ribosomal particle(s)/subunit(s). The subcellular targeting of hNMT was also examined by immunoblotting subcellular fractions from HeLa cells transfected with plasmids containing FLAG epitope-tagged hNMT inserts corresponding either to the originally assigned hNMT gene or to an alternative open reading frame initiated from an in-frame start site upstream from the assumed hNMT start site. Anti-FLAG immunoblotting of cells transfected with a plasmid containing the larger insert revealed FLAG-NMT primarily in the ribosomal fraction with an apparent molecular mass similar to the ≈60-kDa native hNMT. In contrast, immunoblotting of cells transfected with a plasmid containing the smaller insert identified a ≈50-kDa FLAG-NMT predominantly in the cytosolic fraction. An analysis of mixtures of CEM ribosomes and serial dilutions of purified recombinant FLAG-NMTs demonstrated that the ≈60-kDa FLAG-NMT binds ribosomes with higher affinity than the ≈50-kDa FLAG-NMT. These in vivo and in vitrosubcellular targeting and recombinant expression experiments identify a native hNMT that is 10–12 kDa larger than the enzyme predicted by the originally assigned hNMT gene and which is apparently translated from an alternative up-stream start site. The data also indicate that although the unique NH2-terminal residues encoded by this larger open reading frame are not required for in vitrocatalytic activity, they do provide signal(s) involved in targeting hNMT to the ribosomal subcellular fraction where cotranslationalN-myristoylation occurs.