The Yeast Magmas Ortholog Pam16 Has an Essential Function in Fermentative Growth That Involves Sphingolipid Metabolism

Magmas is a growth factor responsive gene encoding an essential mitochondrial protein in mammalian cells. Pam16, the Magmas ortholog in Saccharomyces cerevisiae, is a component of the presequence translocase-associated motor. A temperature-sensitive allele (pam16-I61N) was used to query an array of non-essential gene-deletion strains for synthetic genetic interactions. The pam16-I61N mutation at ambient temperature caused synthetic lethal or sick phenotypes with genes involved in lipid metabolism, perixosome synthesis, histone deacetylation and mitochondrial protein import. The gene deletion array was also screened for suppressors of the pam16-I61N growth defect to identify compensatory pathways. Five suppressor genes were identified (SUR4, ISC1, IPT1, SKN1, and FEN1) and all are involved in sphingolipid metabolism. pam16-I61N cells cultured in glucose at non-permissive temperatures resulted in rapid growth inhibition and G1 cell cycle arrest, but cell viability was maintained. Altered mitochondria morphology, reduced peroxisome induction in glycerol/ethanol and oleate, and changes in the levels of several sphingolipids including C18 alpha-hydroxy-phytoceramide, were also observed in the temperature sensitive strain. Deletion of SUR4, the strongest suppressor, reversed the temperature sensitive fermentative growth defect, the morphological changes and the elevated levels of C18 alpha-hydroxy phytoceramide in pam16-I61N. Deletion of the other four suppressor genes had similar effects on C18 alpha-hydroxy-phytoceramide levels and restored proliferation to the pam16-I61N strain. In addition, pam16-I61N inhibited respiratory growth, likely by reducing cardiolipin, which is essential for mitochondrial function. Our results suggest that the pleiotropic effects caused by impaired Pam16/Magmas function are mediated in part by changes in lipid metabolism.

Magmas是一类生长因子响应基因，其编码哺乳动物细胞内一种必需的线粒体蛋白。Pam16是酿酒酵母（Saccharomyces cerevisiae）中Magmas的直系同源蛋白（ortholog），属于前体序列转位酶相关动力复合物（presequence translocase-associated motor）的组分之一。研究人员利用温度敏感等位基因（temperature-sensitive allele）pam16-I61N，针对非必需基因缺失菌株阵列开展合成遗传相互作用（synthetic genetic interactions）筛选。在常温环境下，pam16-I61N突变会与参与脂质代谢、过氧化物酶体（peroxisome）合成、组蛋白去乙酰化（histone deacetylation）及线粒体蛋白导入（mitochondrial protein import）的基因产生合成致死或生长减损表型。研究团队同时针对该菌株阵列开展了pam16-I61N生长缺陷抑制因子（suppressor）的筛选，以鉴定补偿通路。最终共鉴定出5个抑制基因（SUR4、ISC1、IPT1、SKN1及FEN1），上述基因均参与鞘脂代谢（sphingolipid metabolism）过程。在非允许温度下以葡萄糖为碳源培养的pam16-I61N细胞会快速出现生长抑制与G1期细胞周期阻滞（G1 cell cycle arrest），但仍维持细胞活力（cell viability）。该温度敏感菌株同时表现出线粒体形态改变、在甘油/乙醇及油酸盐（oleate）培养基中过氧化物酶体诱导能力下降，以及包括C18α-羟基植物神经酰胺（C18 alpha-hydroxy-phytoceramide）在内的多种鞘脂水平异常变化。作为最强效的抑制因子，SUR4的缺失能够逆转pam16-I61N的温度敏感发酵生长缺陷、线粒体形态异常及C18α-羟基植物神经酰胺水平升高表型。其余4个抑制基因的缺失同样能够恢复pam16-I61N菌株的增殖能力，并对C18α-羟基植物神经酰胺水平产生类似的调控效果。此外，pam16-I61N突变会抑制呼吸生长，这可能与线粒体功能必需的心磷脂（cardiolipin）水平降低有关。本研究结果表明，Pam16/Magmas功能受损所引发的多效性效应（pleiotropic effects），部分由脂质代谢改变所介导。