Adair W S, Steinmetz S A, Mattson D M, Goodenough U W, Heuser J E
Department of Biology, Washington University, St. Louis, Missouri 63130.
J Cell Biol. 1987 Nov;105(5):2373-82. doi: 10.1083/jcb.105.5.2373.
The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria.
莱茵衣藻的细胞壁由富含羟脯氨酸的糖蛋白组成，排列成五个不同的层。W6（晶体）层包含三种主要糖蛋白（GP1、GP2、GP3），可用离液剂选择性提取，它们在体外可自组装成晶体。开发了一种以定量方式研究W6组装的系统，该系统使用高氯酸盐提取的衣藻细胞作为成核剂。通过FITC-链霉亲和素荧光和快速冷冻/深度蚀刻电子显微镜监测生物素化W6单体的壁重建。在单体浓度（0.2-0.3mg/ml）远低于非成核组装所需浓度时获得了最佳重建。组装从多个成核位点发生，并忠实地反映了完整W6层的结构。使用两个细胞壁突变体（cw-2和cw-15）证明了成核组装的特异性；它们都不作为野生型单体组装的底物。此外，W6亚层由纯化的成分组装而成：GP2和GP3共同组装形成内部（W6A）亚层；然后这作为GP1自组装成外部（W6B）亚层的底物。最后，通过进行种间重建探索了莱茵衣藻与团藻目另外两个成员（衣藻和盘藻）之间的进化关系。在莱茵衣藻和盘藻之间获得了杂交壁，但与衣藻没有，这证实了基于结构标准的分类学归属。
