Myre Michael A, O'Day Danton H
Department of Zoology, University of Toronto at Mississauga, Mississauga, Ontario L5L 1C6, Canada.
J Biol Chem. 2002 May 31;277(22):19735-44. doi: 10.1074/jbc.M109717200. Epub 2002 Mar 27.
Probing of Dictyostelium discoideum cell extracts after SDS-PAGE using (35)S-recombinant calmodulin (CaM) as a probe has revealed approximately three-dozen Ca(2+)-dependent calmodulin binding proteins. Here, we report the molecular cloning, expression, and subcellular localization of a gene encoding a novel calmodulin-binding protein (CaMBP); we have called nucleomorphin, from D. discoideum. A lambdaZAP cDNA expression library of cells from multicellular development was screened using a recombinant calmodulin probe ((35)S-VU1-CaM). The open reading frame of 1119 nucleotides encodes a polypeptide of 340 amino acids with a calculated molecular mass of 38.7 kDa and is constitutively expressed throughout the Dictyostelium life cycle. Nucleomorphin contains a highly acidic glutamic/aspartic acid inverted repeat (DEED) with significant similarity to the conserved nucleoplasmin domain and a putative transmembrane domain in the carboxyl-terminal region. Southern blotting reveals that nucleomorphin exists as a single copy gene. Using gel overlay assays and CaM-agarose we show that bacterially expressed nucleomorphin binds to bovine CaM in a Ca(2+)-dependent manner. Amino-terminal fusion to the green fluorescence protein (GFP) showed that GFP-NumA localized to the nucleus as distinct arc-like patterns similar to heterochromatin regions. GFP-NumA lacking the acidic DEED repeat still showed arc-like accumulations at the nuclear periphery, but the number of nuclei in these cells was increased markedly compared with control cells. Cells expressing GFP-NumA lacking the transmembrane domain localized to the nuclear periphery but did not affect nuclear number or gross morphology. Nucleomorphin is the first nuclear CaMBP to be identified in Dictyostelium. Furthermore, these data present the first identification of a member of the nucleoplasmin family as a calmodulin-binding protein and suggest nucleomorphin has a role in nuclear structure in Dictyostelium.
使用（35）S-重组钙调蛋白（CaM）作为探针，对经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）处理的盘基网柄菌细胞提取物进行检测，结果显示约有三打钙（Ca2+）依赖性钙调蛋白结合蛋白。在此，我们报告了一种编码新型钙调蛋白结合蛋白（CaMBP）基因的分子克隆、表达及亚细胞定位；我们将其命名为核形态蛋白，来自盘基网柄菌。使用重组钙调蛋白探针（（35）S-VU1-CaM）筛选多细胞发育阶段细胞的λZAP cDNA表达文库。1119个核苷酸的开放阅读框编码一个由340个氨基酸组成的多肽，计算分子量为38.7 kDa，在盘基网柄菌的整个生命周期中组成性表达。核形态蛋白含有一个高度酸性的谷氨酸/天冬氨酸反向重复序列（DEED），与保守的核质蛋白结构域具有显著相似性，并且在羧基末端区域有一个推定的跨膜结构域。Southern印迹分析表明核形态蛋白作为单拷贝基因存在。使用凝胶覆盖分析和CaM-琼脂糖，我们发现细菌表达的核形态蛋白以钙（Ca2+）依赖性方式与牛CaM结合。与绿色荧光蛋白（GFP）的氨基末端融合显示，GFP-NumA定位于细胞核，呈类似于异染色质区域的独特弧形模式。缺乏酸性DEED重复序列的GFP-NumA仍在核周边显示弧形聚集，但与对照细胞相比，这些细胞中的细胞核数量明显增加。表达缺乏跨膜结构域的GFP-NumA的细胞定位于核周边，但不影响细胞核数量或总体形态。核形态蛋白是在盘基网柄菌中鉴定出的首个核CaMBP。此外，这些数据首次鉴定出核质蛋白家族的一个成员作为钙调蛋白结合蛋白，并表明核形态蛋白在盘基网柄菌的核结构中起作用。
