Slaninová Iva, Holubárová Alena, Svoboda Augustin
Department of Biology, Faculty of Medicine, Masaryk University, Jostova 10, 66243 Brno, Czech Republic.
Can J Microbiol. 2003 Mar;49(3):189-96. doi: 10.1139/w03-026.
Spectrin, a component of the membrane skeleton in erythrocytes and other animal cells, has also been identified in plant and fungal cells. However, its postulated role, i.e., the maintenance of shape and elasticity of the plasma membrane, is probably not exerted in walled cells. To study spectrin in these cells, we chose yeasts because of a high morphological variability of their life cycle. The localization of spectrin in the cells and protoplasts of Saccharomyces cerevisiae and Schizosaccharomyces japonicus var. versatilis was detected by immunoblotting, indirect immunofluorescence, and immunogold electron microscopy techniques with the use of anti-chicken and anti-human erythrocyte spectrin antibodies. A protein band of 220-240 kDa and some bands of lower relative mass were detected in cell and protoplast extracts of both yeast strains. Spectrin-like proteins were revealed by fluorescence microscopy at cell surfaces and in vacuolar membranes. Immunogold-labelling showed spectrin-like proteins in the plasma membrane, endoplasmic reticulum, vacuoles, nuclei, vesicles, mitochondria, and cell walls. The topology of spectrin was not affected by actin depolymerization with Latrunculin B nor was it changed in either act1-1 or cdc42 mutants, under restrictive conditions. Under osmotic stress, both spectrin and actin were delocalized and appeared in the form of large clusters in the cytoplasm. It is concluded that a protein cross-reacting with spectrin antibodies is present in fission and budding yeasts. Generally, it is located in the proximity of the plasma membrane and other intracellular membranes, probably as a part of the membrane skeleton. No evidence of its relationship to either actin or growth zones of the cell can be provided.
血影蛋白是红细胞和其他动物细胞膜骨架的组成成分，在植物和真菌细胞中也已被鉴定出来。然而，其假定的作用，即维持质膜的形状和弹性，在有细胞壁的细胞中可能并未发挥。为了研究这些细胞中的血影蛋白，我们选择了酵母，因为它们的生命周期具有高度的形态变异性。利用抗鸡和抗人红细胞血影蛋白抗体，通过免疫印迹、间接免疫荧光和免疫金电子显微镜技术检测了酿酒酵母和日本裂殖酵母变种细胞及原生质体中血影蛋白的定位。在两种酵母菌株的细胞和原生质体提取物中均检测到一条220 - 240 kDa的蛋白带以及一些相对分子质量较低的条带。荧光显微镜在细胞表面和液泡膜中发现了血影蛋白样蛋白。免疫金标记显示质膜、内质网、液泡、细胞核、囊泡、线粒体和细胞壁中存在血影蛋白样蛋白。在限制条件下，Latrunculin B使肌动蛋白解聚，这对血影蛋白的拓扑结构没有影响，在act1 - 1或cdc42突变体中也未发生变化。在渗透胁迫下，血影蛋白和肌动蛋白都发生了定位改变，并以大聚集体的形式出现在细胞质中。得出的结论是，裂殖酵母和芽殖酵母中存在一种与血影蛋白抗体发生交叉反应的蛋白。一般来说，它位于质膜和其他细胞内膜附近，可能是膜骨架的一部分。没有证据表明它与肌动蛋白或细胞生长区域存在关联。
