Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria -: Involvement of the Flx1p carrier in FAD export
Riboflavin uptake and FAD synthesis in Saccharomyces cerevisiae mitochondria -: Involvement of the Flx1p carrier in FAD export
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DOI：
10.1074/jbc.m308230200
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发表时间：
2004-01-02
影响因子：
4.8
通讯作者：
Barile, M
中科院分区：
文献类型：
--
作者：
Bafunno, V;Giancaspero, TA;Barile, M
我们研究了酿酒酵母线粒体从胞质核黄素合成FAD的功能步骤。核黄素进入线粒体的摄取是通过与（至少两种）载体系统的存在相一致的机制发生的。FAD在线粒体内由线粒体FAD合成酶（EC www.example.com）合成，并且其经由输出系统输出到胞质溶胶中，该输出系统被光黄素抑制，并且该输出系统不同于核黄素摄取系统。为了了解推定的线粒体FAD载体Flx1p在该途径中的作用，构建了flx1Delta突变株。从flx1Delta突变体细胞分离的耦合线粒体与野生型线粒体进行了比较方面的能力，采取Rf，从它合成FAD，并出口FAD到线粒体外阶段。从flx1Delta突变细胞分离的线粒体特异性地丧失了输出FAD的能力，但没有丧失摄取Rf、FAD或FMN以及从Rf合成FAD的能力。因此，Flx1p被认为是线粒体FAD输出载体。此外，FLX1基因的缺失导致线粒体硫辛酰胺脱氢酶和琥珀酸脱氢酶（FAD结合酶）的活性特异性降低。对于琥珀酸脱氢酶的黄素蛋白亚基，我们可以证明，这不是由于线粒体FAD水平的变化或蛋白质的黄素化程度的变化。相反，琥珀酸脱氢酶的黄素蛋白亚基的量大大减少，表明Flx1p在蛋白质合成或降解中的额外调节作用。
We have studied the functional steps by which Saccharomyces cerevisiae mitochondria can synthesize FAD from cytosolic riboflavin (Rf). Riboflavin uptake into mitochondria took place via a mechanism that is consistent with the existence of ( at least two) carrier systems. FAD was synthesized inside mitochondria by a mitochondrial FAD synthetase (EC 2.7.7.2), and it was exported into the cytosol via an export system that was inhibited by lumiflavin, and which was different from the riboflavin uptake system. To understand the role of the putative mitochondrial FAD carrier, Flx1p, in this pathway, an flx1Delta mutant strain was constructed. Coupled mitochondria isolated from flx1Delta mutant cells were compared with wild-type mitochondria with respect to the capability to take up Rf, to synthesize FAD from it, and to export FAD into the extramitochondrial phase. Mitochondria isolated from flx1Delta mutant cells specifically lost the ability to export FAD, but did not lose the ability to take up Rf, FAD, or FMN and to synthesize FAD from Rf. Hence, Flx1p is proposed to be the mitochondrial FAD export carrier. Moreover, deletion of the FLX1 gene resulted in a specific reduction of the activities of mitochondrial lipoamide dehydrogenase and succinate dehydrogenase, which are FAD-binding enzymes. For the flavoprotein subunit of succinate dehydrogenase we could demonstrate that this was not due to a changed level of mitochondrial FAD or to a change in the degree of flavinylation of the protein. Instead, the amount of the flavoprotein subunit of succinate dehydrogenase was strongly reduced, indicating an additional regulatory role for Flx1p in protein synthesis or degradation.