Kapoor Shobhna, Fansa Eyad K, Möbitz Simone, Ismail Shehab A, Winter Roland, Wittinghofer Alfred, Weise Katrin
Physical Chemistry I - Biophysical Chemistry, TU Dortmund University, Dortmund, Germany; Department of Chemical Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Structural Biology Group, Max Planck Institute of Molecular Physiology, Dortmund, Germany.
Biophys J. 2015 Oct 20;109(8):1619-29. doi: 10.1016/j.bpj.2015.08.033.
The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.
小GTP结合蛋白Arl2和Arl3是紧密的同源物，它们共享许多相互作用的伙伴，并作为异戊二烯化和肉豆蔻酰化货物的置换因子。然而，这两种蛋白具有不同的生物学功能。虽然Arl3被认为是一种纤毛蛋白，但据报道Arl2参与微管蛋白折叠、线粒体功能和Ras信号传导。这两种同源蛋白如何实现这些不同的作用尚未完全了解。最近，我们发现Arl3的N端两亲性螺旋，而不是Arl2的，调节肉豆蔻酰化纤毛蛋白从类GDI溶解因子UNC119a/b的释放。在此呈现的生物物理研究中，两种蛋白均显示在相分离膜的液相无序区域中表现出优先定位和聚集。然而，就其核苷酸负载而言，两种蛋白之间的膜相互作用行为存在显著差异。虽然Arl3和其他具有N端两亲性螺旋的Arf蛋白需要GTP负载才能与膜相互作用，但Arl2以核苷酸非依赖性方式与膜结合。与Arl2相反，Arl3的N端螺旋增加了与UNC119a的结合亲和力。此外，UNC119a阻碍Arl3与膜的结合，但不阻碍Arl2与膜的结合。综上所述，这些结果表明Arl3的核苷酸状态、N端螺旋的位置、膜流动性和结合以及脂质修饰货物从诸如UNC119a的载体中的释放之间存在相互作用。由于假定一种特定的Arl3-GEF存在于纤毛内部，Arl3•GTP的N端螺旋将仅在纤毛内部可用于UNC119a货物释放的变构调节。
