Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles
Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles
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DOI：
10.1523/jneurosci.0447-06.2006
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发表时间：
2006-06-07
影响因子：
5.3
通讯作者：
Gillespie, Peter G.
中科院分区：
文献类型：
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作者：
Hill, Jennifer K.;Williams, Diane E.;Gillespie, Peter G.
在感觉毛细胞中将机械传导定位到毛束需要选择性地将必需蛋白质靶向到特定位置。质膜Ca 2 +-ATP酶（PMCA 2）的亚型2是听觉和平衡所必需的，仅在发束中发现。我们确定了两个主要剪接位点（A和C）的剪接对PMCA 2毛细胞靶向的贡献。当PMCA 2亚型免疫沉淀纯化的大鼠椭圆囊的毛束，2 W是唯一的网站A的变体检测，此外，免疫细胞化学2 W大鼠前庭和耳蜗组织中表明，这种剪接形式仅位于束。为了证明2 w序列的必要性，我们用在剪接位点A和C含有不同变体的PMCA 2转染毛细胞。尽管天然毛束仅在剪接位点C使用2a形式，但表位标记的PMCA 2 w/a和PMCA 2 w/B均集中在毛束中，表明位点C不参与毛束靶向。相比之下，PMCA 2 z/a被排除在束之外，而是靶向基底外侧质膜。用绿色荧光蛋白（GFP）标记的PMCA 2 w/a的束特异性靶向被减弱，表明GFP干扰剪接位点A。总之，这些数据表明，PMCA 2 w/a是大鼠毛细胞中PMCA的毛束同种型，2 w将PMCA 2靶向到毛束。因此，2 w序列是第一个确定的毛束膜蛋白的靶向信号;此外，内耳PMCA异构体的选择性靶向决定的惊人分布表明了分离的Ca 2+转运途径的关键功能作用。
Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites ( A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hair-bundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca2+ transport.