Localization of proteins in yeast by immuno-electron microscopy

酵母作为真核细胞的模型系统已被研究用于遗传分析，但被认为在超微结构研究中前景不佳。近年来，快速冷冻替代法不仅大大提高了酵母细胞超微结构的固定，而且提高了蛋白质的免疫活性，使蛋白质能够相对于超微结构进行定位。我们比较了不同替代方法在免疫电子显微镜下对酵母蛋白超微结构的定位结果。(1)将细胞快速固定在液氮冷却的液态丙烷中，用4%O_sO4/丙酮（-80 C）取代，并包埋在Spurr's树脂中。切片先用抗天冬氨酸蛋白酶抗体处理，再用胶体金偶联抗兔IgG抗体处理。以酿酒酵母为材料，表达了一种丝状真菌天门冬氨酸蛋白酶的异源序列缺失衍生物。免疫电镜分析显示，DELTApro聚集体确实在内质网和核膜中作为电子密集区域可见。(2)细胞快速冷冻后用乙醇替代，在LR White树脂中聚合。切片用抗myc抗体处理，然后用胶体金偶联抗小鼠IgG抗体处理。我们分离了外链糖基化缺陷突变体来研究高尔基体的糖基化机制。抗钒酸盐和未成熟糖基化突变体属于9个互补组。其中一个是VRG4，它编码高尔基gdp -甘露糖转运蛋白（一种跨膜蛋白）。材料为S.cerevisiae，其中myc标记的Vrg4蛋白过量产生。这些结果表明高尔基体膜和内质网膜增生。其中一些膜显示出像哺乳动物细胞一样的堆叠结构。同时，免疫金标记显示过量产生的Vrg4-myc蛋白定位在这种增殖的膜结构中。在这两项研究中，细胞都显示出良好的超微结构保存和蛋白质免疫活性。少

Yeasts have been studied as a model system of eukaryotic cell for the genetic analysis, but regarded not promising for ultrastucture studies. Recently rapid freezing and substitution method has improved greatly the fixation not only of ultrastructure of yeast cell but also of immunoactvity of the proteins, which enabled to localize the proteins relative to the ultrastucture.We compared the results studied by immuno-electron microscopy using different substitution methods to localize the proteins with respect to the ultrastucture in the yeast Saccharomyces cerevisiae.(1) Cells were rapidly fixed into liquid propane cooled with liquid nitrogen and substituted with 4%O_sO4/acetone (-80ﾟC) and embedded in Spurr's resin. Sections were treated with anti aspartic proteinase antibody and then with colloidal gold-conjugated anti-rabbit IgG antibody. The material was Saccharomyces cerevisiae in which heterologous prosequence-deleted derivative of an aspartic proteinase from a filamentous fungus … More Rhizopus niveus was expressed. Immuno-electron microscopical analysis revealed that the DELTApro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope.(2) Cells were substituted with ethanol after rapid frozen and polymerized in LR White resin. Sections were treated with anti-myc antibody and then with colloidal gold-conjugated anti-mouse IgG antibody. We isolated mutants defective in glycosylation of the outer chain to investigate glycosylation mechanism in the Golgi . Vanadate-resistant and immature glycosylation mutants belonged to nine complementation groups. One of them was VRG4 which encoded a Golgi GDP-mannose transporter (a transmembrane protein). The material was S.cerevisiae in which the myc-tagged Vrg4 protein was overproduced. These results showed that the Golgi and ER membranes proliferated. Some of these membranes showed the stacked structures like in mammalian cells. At the same time, immunogold labeling revealed that the overproduced Vrg4-myc proteins localized in such proliferated membrane structures.In both studies, cells were showed a good preservation of ultrastructure and immno activity of proteins. Less