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Volume 272, Number 45, Issue of November 7, 1997 pp. 28680-28689
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, July 30, 1997, and in revised form, August 27, 1997)

From the Pharmacology and Experimental Therapeutic Section, Laboratory of Drug Discovery, Research and Development, Developmental Therapeutic Program, Division of Cancer Treatment, Diagnosis, and Centers, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702

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 exhibiting A₂₆₀/A₂₈₀ 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 vitro subcellular 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 vitro catalytic activity, they do provide signal(s) involved in targeting hNMT to the ribosomal subcellular fraction where cotranslational N-myristoylation occurs.

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