Residues in internal repeats of the rice cation/H+ exchanger are involved in the transport and selection of cations
Residues in internal repeats of the rice cation/H+ exchanger are involved in the transport and selection of cations
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DOI：
10.1074/jbc.m309726200
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发表时间：
2004-01-02
影响因子：
4.8
通讯作者：
Maeshima, M
中科院分区：
文献类型：
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作者：
Kamiya, T;Maeshima, M
在植物中，阳离子/H+交换器（CAX）利用H+-ATP酶和H+-焦磷酸酶形成的H+梯度将Ca 2+和其他金属离子转运到液泡中。这种携带11个跨膜结构域（TM）的交换剂已经从植物、酵母和细菌中分离。在这项研究中，几个CAX的多重序列比对揭示了TM 3和TM 4之间以及TM 8和TM 9之间高度保守的36个残基区域的存在。这两个重复基序被命名为重复序列c-1和c-2。使用定点突变，我们在水稻CAX重复序列中产生了31个突变，这些突变使Ca 2+和Mn 2+发生易位。突变交换器在酿酒酵母菌株中表达，该菌株对Ca 2+和Mn 2+敏感，因为缺乏液泡Ca 2 +-ATP酶和Ca 2 +/H+交换器。根据对Ca ~（2+）和Mn ~（2+）的耐受性，将突变体分为6类。例如，III类突变体对任一离子都没有耐受性，而IV类突变体仅对Ca 2+具有耐受性。对各残留物的生化功能进行了评价。我们使用半胱氨酸诱变和巯基试剂相结合的方法研究了重复序列的膜拓扑结构。我们的研究结果表明，重复c-1重新进入膜从液泡腔侧，并形成一个解决方案可及的区域。此外，重复序列c-1和c-2中的几个残基被发现在动物Na+/Ca 2+交换器中是保守的。最后，我们认为这些重入重复序列可能形成阳离子选择的前庭或过滤器。
In plants, the cation/H+ exchanger (CAX) translocates Ca2+ and other metal ions into vacuoles using the H+ gradient formed by H+-ATPase and H+-pyrophosphatase. Such exchangers carrying 11 transmembrane domains (TMs) have been isolated from plants, yeast, and bacteria. In this study, multiple sequence alignment of several CAXs revealed the presence of highly conserved 36-residue regions between TM3 and TM4 and between TM8 and TM9. These two repetitive motifs are designated repeats c-1 and c-2. Using site-directed mutagenesis, we generated 31 mutations in the repeats of the Oryza sativa CAX, which translocates Ca2+ and Mn2+. Mutant exchangers were expressed in a Saccharomyces cerevisiae strain that is sensitive to Ca2+ and Mn2+ because of the absence of vacuolar Ca2+-ATPase and the Ca2+/H+ exchanger. Mutant exchangers were classified into six classes according to their tolerance for Ca2+ and Mn2+. For example, the class III mutants had no tolerance for either ion, and the class IV mutants had tolerance only for Ca2+. The biochemical function of each residue was estimated. We investigated the membrane topology of the repeats using a method combining cysteine mutagenesis and sulfhydryl reagents. Our results suggest that repeat c-1 re- enters the membrane from the vacuolar luminal side and forms a solution-accessible region. Furthermore, several residues in repeats c-1 and c-2 were found to be conserved in animal Na+/Ca2+ exchangers. Finally, we suggest that these reentrant repeats may form a vestibule or filter for cation selection.