Hedfalk Kristina, Bill Roslyn M, Mullins Jonathan G L, Karlgren Sara, Filipsson Caroline, Bergstrom Johanna, Tamás Markus J, Rydström Jan, Hohmann Stefan
Department of Cell and Molecular Biology/Microbiology, Götegorg University, Götegorg, Sweden.
J Biol Chem. 2004 Apr 9;279(15):14954-60. doi: 10.1074/jbc.M313126200. Epub 2004 Jan 28.
The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydström, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 6337-6345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1Delta strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535-546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane.
酿酒酵母基因FPS1编码主要内在蛋白（MIP）家族的一种水甘油通道蛋白。Fps1p的主要功能似乎是在酵母细胞适应较低外部渗透压时甘油的外流。Fps1p是该家族的一个非典型成员，因为由于两端存在长的亲水性延伸，该蛋白（669个氨基酸）比大多数MIP大得多。我们之前已经表明，该蛋白N端延伸中的一个短结构域对于限制甘油通过通道的运输是必需的（塔马斯，M. J.，卡尔格伦，S.，比尔，R. M.，赫德法尔克，K.，阿莱格里，L.，费雷拉，M.，特维莱因，J. M.，里德斯特伦，J.，穆林斯，J. G. L.，和霍曼，S.（2003年）《生物化学杂志》278卷，6337 - 6345页）。N端结构域的缺失导致通道不受调控、甘油流失和渗透敏感性。在这项工作中，我们研究了Fps1p C端（139个氨基酸）的作用。构建了一组八个截短体，并在酵母fps1Delta菌株中进行了体内测试。我们进行了生长测试、细胞分级分离后的膜定位、甘油积累测量以及渗透应激反应研究。我们的结果表明，C端延伸也参与限制通过Fps1p的运输。我们确定了靠近第六个跨膜结构域的一段12个氨基酸的序列，即535 - 546位残基。这个元件似乎对控制Fps1p功能很重要。与N端结构域类似，C端结构域是两亲性的，有潜在的插入膜中的能力。
