The alkylphosphocholine (APC) class of antineoplastic and antiprotozoal drugs, such as edelfosine and miltefosine, are structural mimics of lyso-phosphatidylcholine (lyso-PC), and are inhibitory to the yeast Saccharomyces cerevisiae at low micromolar concentrations. Cytotoxic effects related to inhibition of phospholipid synthesis, induction of an unfolded protein response, inhibition of oxidative phosphorylation, and disruption of lipid rafts have been attributed to members of this drug class, however the molecular mechanisms of action of these drugs remain incompletely understood. Cytostatic and cytotoxic effects of the alkylphosphocholines exhibit variability with regard to chemical structure, leading to differences in effectiveness against different organisms or cell types. We now report the comprehensive identification of Saccharomyces cerevisiae titratable-essential gene and haploid non-essential gene deletion mutants that are resistant to the APC drug miltefosine (hexadecyl-O-phosphocholine). 58 strains out of ~5600 tested displayed robust and reproducible resistance to miltefosine. This gene set was heavily enriched in functions associated with vesicular transport steps, especially those involving endocytosis and retrograde transport of endosome derived vesicles to the Golgi or vacuole, suggesting a role for these trafficking pathways in transport of miltefosine to potential sites of action in the endoplasmic reticulum (ER) and mitochondrion. In addition, we identified mutants with defects in phosphatidylinositol-4-phosphate synthesis (TetO::STT4) and hydrolysis (sac1∆), an oxysterol binding protein homolog (osh2∆), a number of ER resident proteins, and multiple components of the eisosome. These findings suggest that ER-plasma membrane contact sites and retrograde vesicle transport are involved in the interorganelle transport of lyso-PtdCho and related lyso-phospholipid-like analogs to their intracellular sites of cytotoxic activity.

中文翻译：

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酿酒酵母中的功能基因组筛选揭示了对不同的烷基磷酸胆碱化学治疗剂的耐药性的不同机制。

烷基磷脂酰胆碱（APC）类抗肿瘤药和抗原生动物药物，例如edelfosine和miltefosine，是溶血磷脂酰胆碱（lyso-PC）的结构模拟物，并且在低微摩尔浓度下对酵母酿酒酵母具有抑制作用。与抑制磷脂合成，诱导未折叠的蛋白质反应，抑制氧化磷酸化和破坏脂筏相关的细胞毒性作用已归因于该药物类别，但是这些药物的分子机制尚不完全清楚。烷基磷酸胆碱的细胞生长抑制作用和细胞毒性作用在化学结构方面表现出差异性，导致针对不同生物体或细胞类型的功效差异。我们现在报告对酿酒酵母可滴定的必需基因和对APC药物miltefosine（十六烷基-O-磷酸胆碱）有抗性的单倍体非必需基因缺失突变体的全面鉴定。在〜5600个受测菌株中，有58个菌株显示出对miltefosine的强大且可再现的抗性。该基因集在与囊泡转运步骤有关的功能中高度丰富，尤其是那些涉及胞吞作用和将内体来源的囊泡逆行转运到高尔基体或液泡的功能，提示这些转运途径在将米替福辛转运到内质的潜在作用位点中具有作用网状细胞（ER）和线粒体。此外，我们发现了在磷脂酰肌醇-4-磷酸合成（TetO :: STT4）和水解（sac1∆）中存在缺陷的突变体，氧固醇结合蛋白同源物（osh2∆），许多ER驻留蛋白以及该酶体的多种成分。这些发现表明，ER-质膜接触位点和逆行囊泡转运参与溶血-PtdCho和相关溶血磷脂样类似物的细胞间转运至其细胞毒性活性的细胞内部位。