van de Velde H J, Roebroek A J, Senden N H, Ramaekers F C, Van de Ven W J
Laboratory for Molecular Oncology, University of Leuven, Belgium.
J Cell Sci. 1994 Sep;107 ( Pt 9):2403-16. doi: 10.1242/jcs.107.9.2403.
The novel NSP gene was previously shown to encode, among a variety of neuroendocrine cell types, two 3'-overlapping transcripts, a 3.4 kb one for NSP-A (776 amino acids) and a 1.8 kb one for NSP-C (208 amino acids). The deduced proteins, which were predicted to possess distinct amino-terminal regions, appeared to exhibit some architectural resemblance to known neuroendocrine proteins. In this paper the biochemical characterization and subcellular localization of the two proteins is addressed. In vitro translation of NSP-A and -C RNA produced proteins of about 135 and 23 kDa, respectively. Proteins of similar molecular mass were also detected in immunoprecipitation and western blot analyses of neural and endocrine cells using specific anti-NSP-A or -C antisera; some heterogeneity of NSP-A was observed. NSP-A, but not NSP-C, appeared to be highly phosphorylated and preferentially on serine residues. In immunocytochemical studies, we demonstrated that NSP-A and -C are associated with the endoplasmic reticulum; NSP-A was found to co-localize with SERCA2b, a membrane-associated Ca(2+)-ATPase of the endoplasmic reticulum. In Purkinje cells, we found NSP-immunostaining in the perikaryon, the extensive dendritic tree and the axon, also suggesting association with the smooth endoplasmic reticulum. Biochemical studies of NSP-A provided evidence that NSP-A is strongly associated with microsomal membranes and analysis of deletion mutants of NSP-A revealed that the hydrophobic carboxy-terminal portion of the protein, which is also present in NSP-C, is critical for membrane binding. Through database searches, finally, we found two different NSP-related sequences, one in a sequenced region of human chromosome 19, and the second in a human, pancreatic islet-derived partial cDNA, suggesting that the NSP gene is the prototype of a larger gene family. The results of our studies seem to indicate that the NSP-encoded proteins are novel, membrane-anchored components of the endoplasmic reticulum for which we propose the name reticulons.
先前研究表明，新的NSP基因在多种神经内分泌细胞类型中编码两种3'端重叠的转录本，一种3.4 kb的转录本编码NSP-A（776个氨基酸），另一种1.8 kb的转录本编码NSP-C（208个氨基酸）。推导的蛋白质预计具有不同的氨基末端区域，似乎与已知的神经内分泌蛋白有一些结构上的相似性。本文阐述了这两种蛋白质的生化特性和亚细胞定位。NSP-A和-C RNA的体外翻译分别产生了约135 kDa和23 kDa的蛋白质。在使用特异性抗NSP-A或-C抗血清对神经和内分泌细胞进行免疫沉淀和蛋白质印迹分析时，也检测到了分子量相似的蛋白质；观察到NSP-A存在一些异质性。NSP-A似乎高度磷酸化，且优先在丝氨酸残基上磷酸化，而NSP-C则不然。在免疫细胞化学研究中，我们证明NSP-A和-C与内质网相关；发现NSP-A与SERCA2b共定位，SERCA2b是内质网的一种膜相关Ca(2+)-ATP酶。在浦肯野细胞中，我们在胞体、广泛的树突和轴突中发现了NSP免疫染色，这也表明其与滑面内质网相关。对NSP-A的生化研究表明，NSP-A与微粒体膜紧密相关，对NSP-A缺失突变体的分析表明，该蛋白质的疏水羧基末端部分（也存在于NSP-C中）对于膜结合至关重要。最后，通过数据库搜索，我们发现了两个不同的与NSP相关的序列，一个在人类19号染色体的测序区域，另一个在人胰岛来源的部分cDNA中，这表明NSP基因是一个更大基因家族的原型。我们的研究结果似乎表明，NSP编码的蛋白质是内质网新的膜锚定成分，我们提议将其命名为网质蛋白。
