The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
The Fred Wyszkowski Cancer Research Laboratory, Department of Biology, Technion-Israel Institute of Technology, Haifa 3200003, Israel.
J Proteomics. 2021 May 15;239:104169. doi: 10.1016/j.jprot.2021.104169. Epub 2021 Mar 3.
Folates are essential for nucleotide biosynthesis, amino acid metabolism and cellular proliferation. Following carrier-mediated uptake, folates are polyglutamylated by folylpoly-ɣ-glutamate synthetase (FPGS), resulting in their intracellular retention. FPGS appears as a long isoform, directed to mitochondria via a leader sequence, and a short isoform reported as a soluble cytosolic protein (cFPGS). However, since folates are labile and folate metabolism is compartmentalized, we herein hypothesized that cFPGS is associated with the cytoskeleton, to couple folate uptake and polyglutamylation and channel folate polyglutamates to metabolon compartments. We show that cFPGS is a cytoskeleton-microtubule associated protein: Western blot analysis revealed that endogenous cFPGS is associated with the insoluble cellular fraction, i.e., cytoskeleton and membranes, but not with the cytosol. Mass spectrometry analysis identified the putative cFPGS interactome primarily consisting of microtubule subunits and cytoskeletal motor proteins. Consistently, immunofluorescence microscopy with cytosol-depleted cells demonstrated the association of cFPGS with the cytoskeleton and unconventional myosin-1c. Furthermore, since anti-microtubule, anti-actin cytoskeleton, and coatomer dissociation-inducing agents yielded perinuclear pausing of cFPGS, we propose an actin- and microtubule-dependent transport of cFPGS between the ER-Golgi and the plasma membrane. These novel findings support the coupling of folate transport with polyglutamylation and folate channeling to intracellular metabolon compartments. SIGNIFICANCE: FPGS, an essential enzyme catalyzing intracellular folate polyglutamylation and efficient retention, was described as a soluble cytosolic enzyme in the past 40 years. However, based on the lability of folates and the compartmentalization of folate metabolism and nucleotide biosynthesis, we herein hypothesized that cytoplasmic FPGS is associated with the cytoskeleton, to couple folate transport and polyglutamylation as well as channel folate polyglutamates to biosynthetic metabolon compartments. Indeed, using complementary techniques including Mass-spectrometry proteomics and fluorescence microscopy, we show that cytoplasmic FPGS is associated with the cytoskeleton and unconventional myosin-1c. This novel cytoskeletal localization of cytoplasmic FPGS supports the dynamic channeling of polyglutamylated folates to metabolon compartments to avoid oxidation and intracellular dilution of folates, while enhancing folate-dependent de novo biosynthesis of nucleotides and DNA/protein methylation.
叶酸是核苷酸合成、氨基酸代谢和细胞增殖所必需的。叶酸通过叶酸多聚γ-谷氨酸合成酶（FPGS）介导的载体摄取后，被聚谷氨酸化，从而在细胞内保留。FPGS 表现为一种长的同工型，通过前导序列导向线粒体，以及一种短的同工型，报道为可溶性胞质蛋白（cFPGS）。然而，由于叶酸不稳定，叶酸代谢是区室化的，我们假设 cFPGS 与细胞骨架相关联，以连接叶酸摄取和聚谷氨酸化，并将叶酸聚谷氨酸通道到代谢物区室。我们表明 cFPGS 是一种与微管相关的细胞骨架蛋白：Western blot 分析显示，内源性 cFPGS 与不可溶的细胞部分，即细胞骨架和膜相关，但与细胞质不相关。质谱分析鉴定了 cFPGS 的假定相互作用组，主要由微管亚基和细胞骨架马达蛋白组成。一致地，用细胞质耗尽的细胞进行免疫荧光显微镜显示 cFPGS 与细胞骨架和非典型肌球蛋白-1c 相关联。此外，由于抗微管、抗肌动蛋白细胞骨架和衣壳解离诱导剂导致 cFPGS 在核周暂停，我们提出了 cFPGS 在 ER-Golgi 和质膜之间的肌动蛋白和微管依赖性运输。这些新发现支持了叶酸运输与聚谷氨酸化和叶酸通道化到细胞内代谢物区室的偶联。意义：FPGS，一种催化细胞内叶酸聚谷氨酸化和有效保留的必需酶，在过去的 40 年中被描述为可溶性胞质酶。然而，基于叶酸的不稳定性和叶酸代谢和核苷酸合成的区室化，我们假设细胞质 FPGS 与细胞骨架相关联，以连接叶酸运输和聚谷氨酸化，以及将叶酸聚谷氨酸通道到生物合成代谢物区室。事实上，使用互补技术，包括质谱蛋白质组学和荧光显微镜，我们表明细胞质 FPGS 与细胞骨架和非典型肌球蛋白-1c 相关联。细胞质 FPGS 的这种新型细胞骨架定位支持聚谷氨酸化叶酸动态通道到代谢物区室，以避免叶酸的氧化和细胞内稀释，同时增强叶酸依赖的从头核苷酸和 DNA/蛋白质甲基化合成。
