Cabrera Elisa, González-Montelongo Rafaela, Giraldez Teresa, Alvarez de la Rosa Diego, Siverio José M
Department of Biochemistry and Molecular Biology, Institute of Biomedical Technologies (ITB), Nitrogen Metabolism Group, Universidad de La Laguna, La Laguna, Tenerife, Canarias, Spain.
Eukaryot Cell. 2014 Feb;13(2):267-78. doi: 10.1128/EC.00268-13. Epub 2013 Dec 20.
Some eukaryotes, such as plant and fungi, are capable of utilizing nitrate as the sole nitrogen source. Once transported into the cell, nitrate is reduced to ammonium by the consecutive action of nitrate and nitrite reductase. How nitrate assimilation is balanced with nitrate and nitrite efflux is unknown, as are the proteins involved. The nitrate assimilatory yeast Hansenula polymorpha was used as a model to dissect these efflux systems. We identified the sulfite transporters Ssu1 and Ssu2 as effective nitrate exporters, Ssu2 being quantitatively more important, and we characterize the Nar1 protein as a nitrate/nitrite exporter. The use of strains lacking either SSU2 or NAR1 along with the nitrate reductase gene YNR1 showed that nitrate reductase activity is not required for net nitrate uptake. Growth test experiments indicated that Ssu2 and Nar1 exporters allow yeast to cope with nitrite toxicity. We also have shown that the well-known Saccharomyces cerevisiae sulfite efflux permease Ssu1 is also able to excrete nitrite and nitrate. These results characterize for the first time essential components of the nitrate/nitrite efflux system and their impact on net nitrate uptake and its regulation.
一些真核生物，如植物和真菌，能够利用硝酸盐作为唯一的氮源。一旦硝酸盐被转运到细胞内，它会通过硝酸盐还原酶和亚硝酸还原酶的连续作用被还原为铵。目前尚不清楚硝酸盐同化作用是如何与硝酸盐和亚硝酸盐外流保持平衡的，也不清楚其中涉及的蛋白质。多形汉逊酵母这种进行硝酸盐同化作用的酵母被用作剖析这些外流系统的模型。我们鉴定出亚硫酸盐转运蛋白Ssu1和Ssu2是有效的硝酸盐输出蛋白，其中Ssu2在数量上更为重要，并且我们将Nar1蛋白表征为一种硝酸盐/亚硝酸盐输出蛋白。使用缺乏SSU2或NAR1以及硝酸盐还原酶基因YNR1的菌株进行实验表明，净硝酸盐摄取并不需要硝酸盐还原酶活性。生长测试实验表明，Ssu2和Nar1输出蛋白使酵母能够应对亚硝酸盐毒性。我们还表明，著名的酿酒酵母亚硫酸盐外流通透酶Ssu1也能够排泄亚硝酸盐和硝酸盐。这些结果首次表征了硝酸盐/亚硝酸盐外流系统的关键组成部分及其对净硝酸盐摄取及其调节的影响。
