Study on the development and differentiation of endoplasmic reticulum of yeast from the view point of lipid synthesis.

本研究旨在阐明酿酒酵母内质网发育分化的机制，特别是内质网中主要存在的磷脂合成酶的组织结构。结果表明：(1)采用d -山梨醇密度梯度离心技术，建立了一种温和的亚细胞分离方法。(2)制备了血吸虫磷脂酰丝氨酸合成酶（PSS） n端特异性抗体和磷酸化乙醇胺胞基转移酶特异性抗体。(3)构建了一系列编码PSS和大肠杆菌β -半乳糖苷酶n端区域的融合基因。此外，构建了缺失n端50个氨基酸残基的缺失PSS。从定位分析来看，亲水n端和疏水n端（115 ~ 129）都是PSS正确定位ER所必需的。(4)一个pss突变体含有少量的磷脂酰乙醇胺（PE）。这种双突变体在生长过程中对温度敏感，并改变了内质网结构，这表明PE在适当的内质网形态中是必要的。

The aim of this research is to elucidate the mechanism of development and differentiation of endoplasmic reticulum (ER) of Saccharomyces cerevisiae, especially in relation to the organization of phospholipid synthetic enzymes that reside mainly in ER.Results are as follows,(1) We succeeded in development of a gentle subcellular fractionation method that employs D-sorbitol density gradient centrifugation.(2) We prepared antibodies specific to N-terminaus of phosphatidylserine synthase (PSS) and specific to phosphorylethanolamine cytidylyltransferase of S.cervisiae.(3) A series of fusion genes coding for N-terminal regions of PSS and Escherichia coli beta-galactosidase were constructed. In addition to them, a deletion PSS that lacks N-terminal 50 amino acid residues was constructed. From their localization analysis, both the hydrophilic N-terminal region and a hydrophobic region from 115th to 129th were necessary for the proper ER localization of PSS.(4) A pss ect mutant contained low amaount of phosphatidylethanolamine (PE). This double mutant was temperature-sensitive in growth and had altered ER structures, suggesting the necessity of PE in proper ER morphology.