Bomblies L, Biegelmann E, Döring V, Gerisch G, Krafft-Czepa H, Noegel A A, Schleicher M, Humbel B M
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
J Cell Biol. 1990 Mar;110(3):669-79. doi: 10.1083/jcb.110.3.669.
Developing cells of Dictyostelium discoideum contain crystalline inclusion bodies. The interlattice spaces of the crystals are approximately 11 nm, and their edge dimensions vary in aggregating cells from 0.1 to 0.5 micron. The crystals are enclosed by a membrane with the characteristics of RER. To unravel the nature of the crystals we isolated them under electron microscopical control and purified the two major proteins that cofractionate with the crystals, one of an apparent molecular mass of 69 kD, the other of 56 kD. This latter protein proved to be identical with the protein encoded by the developmentally regulated D2 gene of D. discoideum, as shown by its reactivity with antibodies raised against the bacterially expressed product of a D2 fusion gene. The D2 gene is known to be strictly regulated at the transcript level and to be controlled by cAMP signals. Accordingly, very little of the 56-kD protein was detected in growth phase cells, maximal expression was observed at the aggregation stage, and the expression was stimulated by cAMP pulses. The 69-kD protein is the major constituent of the crystals and is therefore called "crystal protein." This protein is developmentally regulated and accumulates in aggregating cells similar to the D2 protein, but is not, or is only slightly regulated by cAMP pulses. mAbs specific for either the crystal protein or the D2 protein, labeled the intracellular crystals as demonstrated by the use of immunoelectron microscopy. The complete cDNA-derived amino acid sequence of the crystal protein indicates a hydrophobic leader and shows a high degree of sequence similarity with Torpedo acetylcholinesterase and rat lysophospholipase. Because the D2 protein also shows sequence similarities with various esterases, the vesicles filled with crystals of these proteins are named esterosomes.
盘基网柄菌发育中的细胞含有晶体包涵体。晶体的晶格间距约为11纳米，在聚集细胞中其边缘尺寸在0.1至0.5微米之间变化。这些晶体被具有糙面内质网特征的膜所包裹。为了弄清楚晶体的性质，我们在电子显微镜控制下分离出它们，并纯化了与晶体共分级分离的两种主要蛋白质，一种表观分子量为69 kD，另一种为56 kD。后一种蛋白质被证明与盘基网柄菌发育调控的D2基因编码的蛋白质相同，这通过其与针对D2融合基因细菌表达产物产生的抗体的反应性得以证明。已知D2基因在转录水平受到严格调控，并受cAMP信号控制。因此，在生长阶段细胞中检测到的56-kD蛋白质非常少，在聚集阶段观察到最大表达，并且cAMP脉冲可刺激其表达。69-kD蛋白质是晶体的主要成分，因此被称为“晶体蛋白”。这种蛋白质在发育过程中受到调控，并且在聚集细胞中积累，类似于D2蛋白质，但不受cAMP脉冲调控，或仅受到轻微调控。针对晶体蛋白或D2蛋白的单克隆抗体，通过免疫电子显微镜证明可标记细胞内的晶体。晶体蛋白完整的cDNA推导氨基酸序列显示有一个疏水前导序列，并且与电鳐乙酰胆碱酯酶和大鼠溶血磷脂酶有高度的序列相似性。由于D2蛋白也与各种酯酶显示序列相似性，充满这些蛋白质晶体的囊泡被命名为酯质体。
