Kumichel Alexandra, Kapp Katja, Knust Elisabeth
Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr.108, 01307, Dresden, Germany.
Present address: Membrane Traffic and Cell Division, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris, France.
Traffic. 2015 Jun;16(6):604-16. doi: 10.1111/tra.12273. Epub 2015 Apr 14.
The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism.
果蝇的I型跨膜蛋白Crumb是维持顶-基上皮细胞极性所必需的顶端决定因子。Crumb在质膜上的水平至关重要，但其调控方式却知之甚少。在针对Crumb蛋白运输调节因子的遗传筛选中，我们鉴定出了Sar1，它是II型被膜蛋白复合体转运囊泡的核心成分。sar1突变体胚胎显示出Crumb在质膜上的定位减少，这一缺陷类似于在分别缺乏Sec23和Sec24CD的haunted和ghost突变体胚胎中观察到的情况。通过在果蝇施奈德细胞中进行脉冲追踪实验以及基于内切糖苷酶H抗性的蛋白质运输动力学分析，我们在Crumb中鉴定出一个RNKR基序，它有助于内质网的有效输出。鉴定出的基序符合高度保守的双碱性RxKR基序，并介导与Sar1的相互作用。RNKR基序对于果蝇胚胎上皮细胞中转基因编码的Crumb向质膜的转运也是必需的。我们的数据首次表明，双碱性基序作为后生动物中I型质膜蛋白内质网输出的信号。
