Identification of a Novel Family of Nonclassic Yeast Phosphatidylinositol Transfer Proteins Whose Function Modulates Phospholipase D Activity and Sec14p-independent Cell Growth

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24–65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with “bypass-Sec14p” mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

酵母Sec14p是一种磷脂酰肌醇转移蛋白（phosphatidylinositol transfer protein，PITP），其对于高尔基体功能及细胞存活至关重要。本研究对5种与Sec14p一级序列同源性达24%~65%的酵母SFH（Sec Fourteen Homologue，Sec十四同源物）蛋白进行了表征。研究发现，与Sec14p具有64%一级序列同源性的Sfh1p，在体内及体外均无法替代Sec14p发挥功能。然而，与Sec14p一级序列相似性较低的SFH蛋白（即Sfh2p、Sfh3p、Sfh4p及Sfh5p）属于新型磷脂酰肌醇转移蛋白（phosphatidylinositol transfer protein，PITPs），在体外仅表现出磷脂酰肌醇转移活性，而无磷脂酰胆碱转移活性。此外，上调Sfh2p、Sfh4p或Sfh5p的表达，可通过磷脂酶D（phospholipase D，PLD）依赖的方式挽救sec14相关的生长缺陷与分泌缺陷。多项独立实验证据进一步表明，在营养体细胞中，SFH家族PITPs的协同作用是高效激活PLD所必需的。这些证据涵盖两个方面：一是在不依赖Sec14p的细胞生长过程中需要SFH蛋白的协同参与；二是在Sec14p缺陷细胞中实现PLD的最佳激活时也需要SFH蛋白的协同作用。与上述研究结果一致，Sfh2p与PLD共定位于内体区室。实验数据表明，SFH基因编码产物与"绕过Sec14p"突变及PLD存在复杂的相互作用，借此酵母可适应Sec14p核心功能的缺失。本研究拓展了酵母中PITP功能的生理范畴，同时首次在体内证实了特定PITPs在促进PLD激活中的作用。