Zheng Jing, Du Guo-Guang, Matsuda Keiji, Orem Alex, Aguiñaga Sal, Deák Levente, Navarrete Enrique, Madison Laird D, Dallos Peter
Auditory Physiology Laboratory, Department of Communication Sciences and Disorders, Northwestern University, Evanston, IL 60208 USA.
J Cell Sci. 2005 Jul 1;118(Pt 13):2987-96. doi: 10.1242/jcs.02431.
Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cell's membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G/Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestin's function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function.
Prestin是一种独特的分子运动蛋白，表达于哺乳动物耳蜗柯蒂氏器外毛细胞（OHC）的外侧质膜中。据认为，Prestin会经历由细胞膜电位驱动的构象变化。OHC长度的由此产生的改变被假定构成了耳蜗放大器。Prestin是阴离子溶质载体家族26（SCL26A）的成员，但它在电压驱动运动的独特功能方面与其他家族成员不同。由于C末端是该家族中保守性最低的区域，我们用一系列缺失、点突变和嵌合突变体研究了其影响。通过测量非线性电容（NLC）和免疫荧光，在异源表达系统中检测了突变体的功能和细胞表达。每个突变体产生了独特的细胞形态模式混合，根据Prestin在细胞内的位置进行分类。缺失突变体（Del516、Del525、Del630、Del590、Del709、Del719）的数据表明，该蛋白几乎全长（>708个氨基酸）对于正常的Prestin表达和功能是必需的。由于大多数缺失突变消除了质膜靶向，通过在氨基酸515或644处将Prestin与两个最密切相关的SCL26A成员（pendrin和假定的阴离子交换器1）的C末端同源部分融合，构建了嵌合蛋白。这些嵌合蛋白的靶向再次不正确（但方式不同），与简单的截短突变体不同，并且都缺乏功能表型。当两个潜在的基底外侧膜靶向基序发生突变（Y520A/Y526A）时，观察到不完全的质膜表达。我们还表明，一些双点突变（V499G/Y501H）在质膜中完全表达但缺乏NLC。这些不带电荷的氨基酸可能在Prestin的功能中具有尚未揭示的重要作用。总之，这些数据表明C末端中的某些特定序列和单个氨基酸对于正确的细胞分布和功能是必需的。
