Kwon Hye-Kyoung, Yokoyama Ryusuke, Nishitani Kazuhiko
Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, 980-8578 Japan.
Plant Cell Physiol. 2005 Jun;46(6):843-57. doi: 10.1093/pcp/pci089. Epub 2005 Mar 15.
To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.
为阐明细胞壁构建的机制，我们采用蛋白质组学方法，研究拟南芥悬浮培养细胞原生质体细胞壁再生过程中分泌到细胞壁间隙的蛋白质。我们聚焦于与细胞壁结构松散结合且能用1 M KCl溶液从以下材料中提取的细胞壁蛋白：（i）天然悬浮培养细胞；（ii）已让其细胞壁再生1小时的原生质体；以及（iii）已让其细胞壁再生3小时的原生质体。我们采用了一种不破坏细胞完整性的非破坏性提取程序，从而避免了细胞质蛋白的污染。使用二维聚丙烯酰胺凝胶电泳（2-D PAGE）和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF/MS），我们分别分离、定位并鉴定了71种源自天然细胞壁的蛋白质，以及分别源自1小时和3小时再生原生质体的175种和212种蛋白质。在这三个蛋白质组分中鉴定出了具有不同翻译后修饰状态（包括磷酸化和糖基化）的截然不同的蛋白质组。这表明原生质体细胞壁再生过程中细胞壁蛋白在细胞壁内发生了动态变化。该分析揭示了一组专门参与悬浮培养细胞中细胞壁扩展和构建的酶。这种方法还确定了一组原本未被预测定位于细胞壁间隙的细胞壁蛋白。
