Kloetzel John A, Baroin-Tourancheau Anne, Miceli Cristina, Barchetta Sabrina, Farmar James, Banerjee Deben, Fleury-Aubusson Anne
Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD 21250, USA.
J Cell Sci. 2003 Apr 1;116(Pt 7):1291-303. doi: 10.1242/jcs.00333.
Protistan cells employ a wide variety of strategies to reinforce and give pattern to their outermost cortical layers. Whereas some use common cytoskeletal elements such as microtubules, others are based on novel cytoskeletal proteins that are as-yet-unknown in higher eukaryotes. The hypotrich ciliate Euplotes possesses a continuous monolayer of scales or plates, located within flattened membranous sacs ('alveoli') just below the plasma membrane, and this provides rigidity and form to the cell. Using immunological techniques, the major proteins comprising these 'alveolar plates' have been identified and termed alpha-, beta-, and gamma-plateins. The present report describes work leading to the molecular characterization of three plateins, alpha 1 and alpha 2 (predicted M(r)s of 61 and 56 kDa) and a beta/gamma form (M(r)=73 kDa). All three proteins have features that are hallmarks of articulins, a class of cytoskeletal proteins that has been identified in the cortex of a wide variety of protistan cells, including certain flagellates, ciliates, dinoflagellates and PLASMODIUM: Chief among these common features are a prominent primary domain of tandem 12-amino acid repeats, rich in valine and proline, and a secondary domain of fewer, shorter repeating units. However, variations in amino acid use within both primary and secondary repetitive domains, and a much more acidic character (predicted pIs of 4.7-4.9), indicate that the plateins represent the first proteins in a new subclass or family of articulins. This conclusion is supported by another novel feature of the plateins, the presence of a canonical hydrophobic signal peptide at the N-terminus of each derived platein sequence. This correlates well with the final cellular location of the plateins, which are assembled into plates within the membrane-limited alveolar sacs. To our knowledge, this is the first report in any eukaryote of cytoskeletal proteins with such start-transfer sequences. Confocal immunofluorescence microscopy, using antibodies to the plateins as probes, reveals that new alveolar plates (enlarging in cortical zones undergoing morphogenesis) label more faintly than mature parental plates. During plate assembly (or polymerization), the plateins thus appear to exist in a more soluble form.
原生生物细胞采用多种策略来强化其最外层皮质层并赋予其形态。一些细胞利用常见的细胞骨架成分，如微管，而另一些则基于高等真核生物中尚未发现的新型细胞骨架蛋白。下毛目纤毛虫游仆虫拥有一层连续的鳞片或板层，位于紧邻质膜下方的扁平膜囊（“泡囊”）内，这为细胞提供了刚性和形态。利用免疫技术，已鉴定出构成这些“泡囊板”的主要蛋白质，并将其命名为α -、β - 和γ - 板蛋白。本报告描述了对三种板蛋白α1和α2（预测分子量分别为61 kDa和56 kDa）以及一种β/γ形式（分子量 = 73 kDa）进行分子表征的工作。这三种蛋白质都具有关节蛋白的特征，关节蛋白是一类在多种原生生物细胞的皮质中已被鉴定出的细胞骨架蛋白，包括某些鞭毛虫、纤毛虫、甲藻和疟原虫：这些共同特征中最主要的是一个突出的由12个氨基酸串联重复组成的一级结构域，富含缬氨酸和脯氨酸，以及一个由较少、较短重复单元组成的二级结构域。然而，一级和二级重复结构域内氨基酸使用的差异，以及更强的酸性特征（预测的pI值为4.7 - 4.9），表明板蛋白代表了关节蛋白新亚类或新家族中的首批蛋白质。板蛋白的另一个新特征支持了这一结论，即在每个推导的板蛋白序列的N端存在一个典型的疏水信号肽。这与板蛋白最终的细胞定位很好地相关，板蛋白在膜限定的泡囊内组装成板层。据我们所知，这是在任何真核生物中首次报道具有这种起始转移序列的细胞骨架蛋白。使用针对板蛋白的抗体作为探针进行共聚焦免疫荧光显微镜观察发现，新的泡囊板（在经历形态发生的皮质区域中增大）的标记比成熟的亲代板更淡。在板层组装（或聚合）过程中，板蛋白因此似乎以更可溶的形式存在。
