Saita Shotaro, Shirane Michiko, Natume Tohru, Iemura Shun-Ichiro, Nakayama Keiichi I
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582, Japan; CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan.
National Institutes of Advanced Industrial Science, Kohtoh-ku, Tokyo 135-0064, Japan.
J Biol Chem. 2009 May 15;284(20):13766-13777. doi: 10.1074/jbc.M807938200. Epub 2009 Mar 16.
Protrudin is a protein that contains a Rab11-binding domain and a FYVE (lipid-binding) domain and that functions to promote neurite formation through interaction with the GDP-bound form of Rab11. Protrudin also contains a short sequence motif designated FFAT (two phenylalanines in an acidic tract), which in other proteins has been shown to mediate binding to vesicle-associated membrane protein-associated protein (VAP). We now show that protrudin associates and colocalizes with VAP-A, an isoform of VAP expressed in the endoplasmic reticulum. Both the interaction between protrudin and VAP-A as well as the induction of process formation by protrudin were markedly inhibited by mutation of the FFAT motif. Furthermore, depletion of VAP-A by RNA interference resulted in mislocalization of protrudin as well as in inhibition of neurite outgrowth induced by nerve growth factor in rat pheochromocytoma PC12 cells. These defects resulting from depletion of endogenous rat VAP-A in PC12 cells were corrected by forced expression of (RNA interference-resistant) human VAP-A but not by VAP-A mutants that have lost the ability to interact with protrudin. These results suggest that VAP-A is an important regulator both of the subcellular localization of protrudin and of its ability to stimulate neurite outgrowth.
Protrudin是一种蛋白质，它含有一个Rab11结合结构域和一个FYVE（脂质结合）结构域，其功能是通过与GDP结合形式的Rab11相互作用来促进神经突形成。Protrudin还含有一个被称为FFAT（酸性区域中的两个苯丙氨酸）的短序列基序，在其他蛋白质中已证明该基序可介导与囊泡相关膜蛋白相关蛋白（VAP）的结合。我们现在表明，Protrudin与VAP-A相互作用并共定位，VAP-A是在内质网中表达的VAP的一种同工型。Protrudin与VAP-A之间的相互作用以及Protrudin诱导的突起形成均被FFAT基序的突变显著抑制。此外，RNA干扰使VAP-A缺失导致Protrudin定位错误，并抑制了大鼠嗜铬细胞瘤PC12细胞中神经生长因子诱导的神经突生长。PC12细胞中内源性大鼠VAP-A缺失导致的这些缺陷，通过强制表达（抗RNA干扰的）人VAP-A得以纠正，但不能通过已失去与Protrudin相互作用能力的VAP-A突变体纠正。这些结果表明，VAP-A既是Protrudin亚细胞定位的重要调节因子，也是其刺激神经突生长能力的重要调节因子。
