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, Maryland 21250, USA.
J Eukaryot Microbiol. 2003 Jan-Feb;50(1):19-33. doi: 10.1111/j.1550-7408.2003.tb00102.x.
In euplotid ciliates, the cortex is reinforced by alveolar plates--proteinaceous scales located within the membranous alveolar sacs, forming a monolayer just below the plasma membrane. This system appears to play a cytoskeletal role analogous to that provided by the fibrous epiplasm found beneath the cortical alveoli in other ciliates. In Euplotes aediculatus, the major alveolar plate proteins (termed alpha-, beta-, and gamma-plateins) have been identified. Using anti-platein antibodies, an expression library of Euplotes genes was screened, and a platein gene identified, cloned, and completely sequenced. Comparison of its derived amino acid sequence with microsequences obtained directly from purified plateins identified this gene as encoding one of the closely related beta- or gamma-plateins. The derived protein, of 644 amino acids (74.9 kDa), is very acidic (pI = 4.88). Microsequences from authentic alpha-platein were then used to design oligonucleotide primers, which yielded, via a PCR-based approach, the sequences of two alpha-platein genes from E. aediculatus. Even more acidic proteins, the derived alpha1- and alpha2-plateins contain 536 and 501 residues, respectively. Analyses of their amino acid sequences revealed the plateins to be members of the articulin superfamily of cytoskeletal proteins, first described in Euglena and now identified in the ciliate Pseudomicrothorax and in Plasmodium. The hallmark articulin repetitive motifs (based on degenerate valine- and proline-rich 12-mers) are present in all three plateins. In beta/gamma-platein this primary motif domain (27 repeats) is central in the molecule, whereas the primary repeats in the alpha-plateins lie near their C-termini. A cluster of proline-rich pentameric secondary repeats is found in the C-terminus of beta/gamma-platein, but near the N-terminus of alpha-plateins. All three plateins contain canonical N-terminal signal sequences, unique among known cytoskeletal proteins. The presence of start-transfer sequences correlates well with the final intra-alveolar location of these proteins. This feature, and significant differences from known articulins in amino acid usage and arrangement within the repeat domains, lead us to propose that the plateins comprise a new family of articulin-related proteins. Efforts to follow microscopically the assembly of plateins into new alveolar plates during pre-fission morphogenesis are underway.
在真核纤毛虫中，皮层由泡状板加强——泡状板是位于膜状泡囊中、在质膜下方形成单层的蛋白质鳞片。该系统似乎起着类似于其他纤毛虫皮层泡囊下方发现的纤维状表质所提供的细胞骨架作用。在尖棘真核虫中，主要的泡状板蛋白（称为α-、β-和γ-板蛋白）已被鉴定。利用抗板蛋白抗体，筛选了真核虫基因的表达文库，并鉴定、克隆和完全测序了一个板蛋白基因。将其推导的氨基酸序列与直接从纯化的板蛋白获得的微序列进行比较，确定该基因编码密切相关的β-或γ-板蛋白之一。推导的蛋白质由644个氨基酸组成（74.9 kDa），酸性很强（pI = 4.88）。然后，利用来自真实α-板蛋白的微序列设计寡核苷酸引物，通过基于PCR的方法获得了尖棘真核虫两个α-板蛋白基因的序列。α1-和α2-板蛋白是酸性更强的蛋白质，分别含有536和501个残基。对它们氨基酸序列的分析表明，板蛋白是细胞骨架蛋白关节蛋白超家族的成员，该超家族最初在眼虫中被描述，现在在纤毛虫伪微胸虫和疟原虫中也有发现。所有三种板蛋白都存在标志性的关节蛋白重复基序（基于富含缬氨酸和脯氨酸的简并12聚体）。在β/γ-板蛋白中，这个主要基序结构域（27个重复）位于分子中心，而α-板蛋白中的主要重复序列位于其C端附近。在β/γ-板蛋白的C端发现了一组富含脯氨酸的五聚体二级重复序列，但在α-板蛋白的N端附近。所有三种板蛋白都含有典型的N端信号序列，这在已知的细胞骨架蛋白中是独一无二的。起始转移序列的存在与这些蛋白质最终在泡囊内的位置密切相关。这一特征，以及在重复结构域内氨基酸使用和排列上与已知关节蛋白的显著差异，使我们提出板蛋白构成了一个新的与关节蛋白相关的蛋白质家族。目前正在努力通过显微镜观察在裂前形态发生过程中板蛋白组装成新的泡状板的过程。
