Caplan S, Green R, Rocco J, Kurjan J
Department of Biological Sciences, Columbia University, New York, New York 10027.
J Bacteriol. 1991 Jan;173(2):627-35. doi: 10.1128/jb.173.2.627-635.1991.
The MF alpha 1 gene encodes a precursor, prepro-alpha-factor, that undergoes several proteolytic processing steps within the classical secretory pathway to produce the mature peptide pheromone, alpha-factor. To investigate the role of structural features of the MF alpha 1 precursor in alpha-factor production, we analyzed the effect of mf alpha 1 mutations that alter precursor structure in a number of ways. These mutations resulted in decreased alpha-factor secretion and intracellular accumulation of pro-alpha-factor. With the exception of the mutant lacking all three N glycosylation sites, the pro-alpha-factor forms that accumulated were core glycosylated but had not yet undergone the addition of outer chain carbohydrate. The delay, therefore, occurred at a step prior to the first proteolytic processing step involved in maturation of the precursor and was probably due to inefficient endoplasmic reticulum-to-Golgi transport. Elimination of all three N-glycosylation sites caused a delay in disappearance of intracellular precursor, and alpha-factor secretion was also slowed. These data indicate that N glycosylation is important but not essential for transport of the precursor through the secretory pathway. The decreased alpha-factor secretion and increased precursor accumulation seen with many different structural changes of pro-alpha-factor indicate that the secretory pathway is extremely sensitive to changes in precursor structure. This sensitivity could cause inefficient secretion of heterologous proteins and hybrids between MF alpha 1 and heterologous proteins in yeast cells.
MFα1基因编码一种前体，即前原α因子，它在经典分泌途径中经历多个蛋白水解加工步骤，以产生成熟的肽信息素α因子。为了研究MFα1前体的结构特征在α因子产生中的作用，我们分析了以多种方式改变前体结构的mfα1突变的影响。这些突变导致α因子分泌减少和前α因子在细胞内积累。除了缺乏所有三个N糖基化位点的突变体外，积累的前α因子形式是核心糖基化的，但尚未进行外链碳水化合物的添加。因此，延迟发生在参与前体成熟的第一个蛋白水解加工步骤之前的一个步骤，可能是由于内质网到高尔基体的运输效率低下。消除所有三个N糖基化位点导致细胞内前体消失延迟，α因子分泌也减慢。这些数据表明，N糖基化对于前体通过分泌途径的运输很重要，但不是必需的。前α因子许多不同结构变化导致α因子分泌减少和前体积累增加，这表明分泌途径对前体结构变化极其敏感。这种敏感性可能导致酵母细胞中异源蛋白以及MFα1与异源蛋白之间的杂种分泌效率低下。
