Metcalf Talibah, van der Wel Hanke, Escalante Ricardo, Sastre Leandro, West Christopher M
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
Eukaryot Cell. 2007 Jul;6(7):1137-49. doi: 10.1128/EC.00329-06. Epub 2007 Apr 6.
Like the cyst walls of other protists, the spore coat of Dictyostelium discoideum is formed de novo to protect the enclosed dormant cell from stress. Spore coat assembly is initiated by exocytosis of protein and polysaccharide precursors at the cell surface, followed by the infusion of nascent cellulose fibrils, resulting in an asymmetrical trilaminar sandwich with cellulose filling the middle layer. A molecular complex consisting of cellulose and two proteins, SP85 and SP65, is associated with the inner and middle layers and is required for proper organization of distinct proteins in the outer layer. Here we show that, unlike SP85 and other protein precursors, which are stored in prespore vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the SP65 locus with green fluorescent protein, we find that SP65 is delivered to the cell surface via largely distinct vesicles, suggesting that separate delivery of components of the cellulose-SP85-SP65 complex regulates its formation at the cell surface. In support of previous in vivo studies, recombinant SP65 and SP85 are shown to interact directly. In addition, truncation of SP65 causes a defect of the outer layer permeability barrier as seen previously for SP85 mutants. These observations suggest that assembly of the cellulose-SP85-SP65 triad at the cell surface is biosynthetically regulated both temporally and spatially and that the complex contributes an essential function to outer layer architecture and function.
与其他原生生物的囊壁一样，盘基网柄菌的孢子壁是重新形成的，以保护被包裹的休眠细胞免受压力。孢子壁的组装始于蛋白质和多糖前体在细胞表面的胞吐作用，随后是新生纤维素微纤丝的注入，形成一个不对称的三层夹心结构，中间层填充着纤维素。由纤维素以及两种蛋白质SP85和SP65组成的分子复合物与内层和中间层相关联，并且是外层中不同蛋白质正确组织所必需的。在这里我们表明，与储存在前孢子囊泡中的SP85和其他蛋白质前体不同，SP65像纤维素一样是及时合成的。通过用绿色荧光蛋白标记SP65基因座，我们发现SP65通过基本上不同的囊泡被递送到细胞表面，这表明纤维素-SP85-SP65复合物各组分的分开递送调节了其在细胞表面的形成。为支持先前的体内研究，重组SP65和SP85被证明可直接相互作用。此外，如先前在SP85突变体中所见，SP65的截短会导致外层渗透屏障的缺陷。这些观察结果表明，纤维素-SP85-SP65三联体在细胞表面的组装在时间和空间上受到生物合成调控，并且该复合物对外层结构和功能起着至关重要的作用。
