Todisco Simona, Di Noia Maria Antonietta, Castegna Alessandra, Lasorsa Francesco Massimo, Paradies Eleonora, Palmieri Ferdinando
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari, via Orabona 4, 70125 Bari, Italy; Center of Excellence in Comparative Genomics, University of Bari, Italy.
Department of Sciences, University of Basilicata, via N. Sauro 85, 85100 Potenza, Italy.
Biochim Biophys Acta. 2014 Feb;1837(2):326-34. doi: 10.1016/j.bbabio.2013.11.013. Epub 2013 Dec 1.
The genome of Saccharomyces cerevisiae contains 35 members of the mitochondrial carrier family, nearly all of which have been functionally characterized. In this study, the identification of the mitochondrial carrier for adenosine 5'-phosphosulfate (APS) is described. The corresponding gene (YPR011c) was overexpressed in bacteria. The purified protein was reconstituted into phospholipid vesicles and its transport properties and kinetic parameters were characterized. It transported APS, 3'-phospho-adenosine 5'-phosphosulfate, sulfate and phosphate almost exclusively by a counter-exchange mechanism. Transport was saturable and inhibited by bongkrekic acid and other inhibitors. To investigate the physiological significance of this carrier in S. cerevisiae, mutants were subjected to thermal shock at 45°C in the presence of sulfate and in the absence of methionine. At 45°C cells lacking YPR011c, engineered cells (in which APS is produced only in mitochondria) and more so the latter cells, in which the exit of mitochondrial APS is prevented by the absence of YPR011cp, were less thermotolerant. Moreover, at the same temperature all these cells contained less methionine and total glutathione than wild-type cells. Our results show that S. cerevisiae mitochondria are equipped with a transporter for APS and that YPR011cp-mediated mitochondrial transport of APS occurs in S. cerevisiae under thermal stress conditions.
酿酒酵母的基因组包含35个线粒体载体家族成员,几乎所有成员的功能都已得到表征。在本研究中,描述了5'-磷酸腺苷硫酸酯(APS)线粒体载体的鉴定。相应基因(YPR011c)在细菌中过表达。纯化的蛋白质被重组成磷脂囊泡,并对其转运特性和动力学参数进行了表征。它几乎完全通过反向交换机制转运APS、3'-磷酸腺苷5'-磷酸硫酸酯、硫酸盐和磷酸盐。转运是可饱和的,并受到邦克雷酸和其他抑制剂的抑制。为了研究这种载体在酿酒酵母中的生理意义,对突变体在存在硫酸盐和不存在甲硫氨酸的情况下于45°C进行热激处理。在45°C时,缺乏YPR011c的细胞、工程细胞(其中APS仅在线粒体中产生),尤其是后者细胞(其中由于缺乏YPR011cp而阻止了线粒体APS的输出)的耐热性较差。此外,在相同温度下,所有这些细胞中的甲硫氨酸和总谷胱甘肽含量均低于野生型细胞。我们的结果表明,酿酒酵母线粒体配备有APS转运体,并且在热应激条件下,YPR011cp介导的APS线粒体转运在酿酒酵母中发生。