Taschner Michael, Kotsis Fruzsina, Braeuer Philipp, Kuehn E Wolfgang, Lorentzen Esben
Department of Structural Cell Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried, Germany.
Renal Division, University Hospital Freiburg, D-79106 Freiburg, Germany.
J Cell Biol. 2014 Oct 27;207(2):269-82. doi: 10.1083/jcb.201408002.
Cilia are microtubule-based organelles that assemble via intraflagellar transport (IFT) and function as signaling hubs on eukaryotic cells. IFT relies on molecular motors and IFT complexes that mediate the contacts with ciliary cargo. To elucidate the architecture of the IFT-B complex, we reconstituted and purified the nonameric IFT-B core from Chlamydomonas reinhardtii and determined the crystal structures of C. reinhardtii IFT70/52 and Tetrahymena IFT52/46 subcomplexes. The 2.5-Å resolution IFT70/52 structure shows that IFT52330-370 is buried deeply within the IFT70 tetratricopeptide repeat superhelix. Furthermore, the polycystic kidney disease protein IFT88 binds IFT52281-329 in a complex that interacts directly with IFT70/IFT52330-381 in trans. The structure of IFT52C/IFT46C was solved at 2.3 Å resolution, and we show that it is essential for IFT-B core integrity by mediating interaction between IFT88/70/52/46 and IFT81/74/27/25/22 subcomplexes. Consistent with this, overexpression of mammalian IFT52C in MDCK cells is dominant-negative and causes IFT protein mislocalization and disrupted ciliogenesis. These data further rationalize several ciliogenesis phenotypes of IFT mutant strains.
纤毛是以微管为基础的细胞器，通过鞭毛内运输（IFT）组装而成，并作为真核细胞上的信号枢纽发挥作用。IFT依赖于分子马达和IFT复合体，后者介导与纤毛货物的接触。为了阐明IFT-B复合体的结构，我们从莱茵衣藻中重组并纯化了九聚体IFT-B核心，并确定了莱茵衣藻IFT70/52和嗜热四膜虫IFT52/46亚复合体的晶体结构。分辨率为2.5埃的IFT70/52结构表明，IFT52330 - 370深深埋在IFT70四肽重复超螺旋内。此外，多囊肾病蛋白IFT88在一个复合体中与IFT52281 - 329结合，该复合体与IFT70/IFT52330 - 381发生反式直接相互作用。IFT52C/IFT46C的结构在2.3埃分辨率下得到解析，我们发现它通过介导IFT88/70/52/46与IFT81/74/27/25/22亚复合体之间的相互作用，对IFT-B核心的完整性至关重要。与此一致的是，在MDCK细胞中过表达哺乳动物IFT52C具有显性负效应，会导致IFT蛋白定位错误并破坏纤毛发生。这些数据进一步解释了IFT突变株的几种纤毛发生表型。
