Munn A L, Heese-Peck A, Stevenson B J, Pichler H, Riezman H
Biozentrum of the University of Basel, CH-4056 Basel, Switzerland.
Mol Biol Cell. 1999 Nov;10(11):3943-57. doi: 10.1091/mbc.10.11.3943.
Sterols are major components of the plasma membrane, but their functions in this membrane are not well understood. We isolated a mutant defective in the internalization step of endocytosis in a gene (ERG2) encoding a C-8 sterol isomerase that acts in the late part of the ergosterol biosynthetic pathway. In the absence of Erg2p, yeast cells accumulate sterols structurally different from ergosterol, which is the major sterol in wild-type yeast. To investigate the structural requirements of ergosterol for endocytosis in more detail, several erg mutants (erg2Delta, erg6Delta, and erg2Deltaerg6Delta) were made. Analysis of fluid phase and receptor-mediated endocytosis indicates that changes in the sterol composition lead to a defect in the internalization step. Vesicle formation and fusion along the secretory pathway were not strongly affected in the ergDelta mutants. The severity of the endocytic defect correlates with changes in sterol structure and with the abundance of specific sterols in the ergDelta mutants. Desaturation of the B ring of the sterol molecules is important for the internalization step. A single desaturation at C-8,9 was not sufficient to support internalization at 37 degrees C whereas two double bonds, either at C-5,6 and C-7,8 or at C-5,6 and C-8,9, allowed internalization.
甾醇是质膜的主要成分，但其在该膜中的功能尚未完全明确。我们在编码C-8甾醇异构酶的基因（ERG2）中分离出一个在内吞作用内化步骤存在缺陷的突变体，该酶在麦角甾醇生物合成途径的后期发挥作用。在缺乏Erg2p的情况下，酵母细胞积累的甾醇在结构上不同于野生型酵母中的主要甾醇麦角甾醇。为了更详细地研究麦角甾醇对胞吞作用的结构要求，构建了几个erg突变体（erg2Δ、erg6Δ和erg2Δerg6Δ）。对液相和受体介导的内吞作用的分析表明，甾醇组成的变化导致内化步骤出现缺陷。ergΔ突变体中沿分泌途径的囊泡形成和融合未受到强烈影响。内吞缺陷的严重程度与甾醇结构的变化以及ergΔ突变体中特定甾醇的丰度相关。甾醇分子B环的去饱和作用对于内化步骤很重要。在C-8,9处的单个去饱和不足以支持在37℃下的内化，而在C-5,6和C-7,8或C-5,6和C-8,9处的两个双键则允许内化。
