Glover C J, Hartman K D, Felsted R L
Developmental Therapeutic Program, Division of Cancer Treatment, Diagnosis, and Centers, NCI-Frederick Cancer Research and Development Center, National Institutes of Health, Frederick, Maryland 21702, USA.
J Biol Chem. 1997 Nov 7;272(45):28680-9. doi: 10.1074/jbc.272.45.28680.
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 approximately 60-kDa protein that was specifically immunoblotted with an anti-human NMT (hNMT) peptide antibody. This approximately 60-kDa protein was stable in the presence of proteolytic enzyme inhibitors but was gradually converted into a approximately 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 approximately 60-kDa native hNMT. In contrast, immunoblotting of cells transfected with a plasmid containing the smaller insert identified a approximately 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 approximately 60-kDa FLAG-NMT binds ribosomes with higher affinity than the approximately 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.
N-肉豆蔻酰基转移酶（NMT）催化多种细胞和病毒蛋白的NH2末端甘氨酸与肉豆蔻酸的共翻译酰化反应。通过差速离心法显示，人淋巴细胞白血病（即CEM和MOLT-4）和子宫颈癌（即HeLa）细胞的等渗细胞匀浆中的大多数体外NMT活性（60-85%）与核糖体亚细胞组分相关。在核糖体组分中还发现了一种约60 kDa的蛋白质，它能与抗人NMT（hNMT）肽抗体发生特异性免疫印迹反应。这种约60 kDa的蛋白质在存在蛋白酶抑制剂的情况下是稳定的，但在没有蛋白酶抑制剂的情况下储存时会逐渐转化为约46 kDa的物种。核糖体组分的蔗糖密度梯度离心导致hNMT活性的沉降与260 nm吸收曲线完全一致，并且A260/A280吸收比值>1.8，表明NMT与假定的核糖体颗粒/亚基相关。还通过对用含有FLAG表位标签的hNMT插入片段的质粒转染的HeLa细胞的亚细胞组分进行免疫印迹分析，研究了hNMT的亚细胞定位，这些插入片段对应于最初指定的hNMT基因或从假定的hNMT起始位点上游的框内起始位点起始的另一个开放阅读框。用含有较大插入片段的质粒转染的细胞的抗FLAG免疫印迹显示，FLAG-NMT主要存在于核糖体组分中，其表观分子量与约60 kDa的天然hNMT相似。相反，用含有较小插入片段的质粒转染的细胞的免疫印迹鉴定出一种约50 kDa的FLAG-NMT主要存在于细胞质组分中。对CEM核糖体与纯化的重组FLAG-NMT系列稀释液的混合物进行分析表明，约60 kDa的FLAG-NMT比约50 kDa的FLAG-NMT以更高的亲和力结合核糖体。这些体内和体外亚细胞定位及重组表达实验鉴定出一种天然hNMT，其比最初指定的hNMT基因预测的酶大10-12 kDa，并且显然是从一个替代的上游起始位点翻译而来。数据还表明虽然这个较大的开放阅读框编码的独特NH2末端残基对于体外催化活性不是必需的，但它们确实提供了参与将hNMT靶向到共翻译N-肉豆蔻酰化发生的核糖体亚细胞组分的信号。
