Safi Frozan, Shteiman-Kotler Alina, Zhong Yunan, Iliadi Konstantin G, Boulianne Gabrielle L, Rotin Daniela
Hospital for Sick Children, Toronto, ON M5G 0A4, Canada Biochemistry Department, University of Toronto, Toronto ON M5S 1A1, Canada.
Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.
Mol Biol Cell. 2016 Mar 15;27(6):907-18. doi: 10.1091/mbc.E15-06-0420. Epub 2016 Jan 28.
Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.
果蝇Nedd4（dNedd4）是一种HECT泛素连接酶，有两种主要的剪接异构体：dNedd4-短型（dNedd4S）和-长型（dNedd4Lo）。DNedd4Lo有一个独特的N端，包含一个富含脯氨酸的区域。我们之前发现，dNedd4S促进神经肌肉突触形成，而dNedd4Lo则抑制它并损害幼虫运动。为了阐明运动受损的原因，我们使用质谱法在幼虫裂解物中寻找与dNedd4Lo的N端独特区域结合的伙伴，并鉴定出了发动蛋白（dAmph）。dAmph是一种含有SH3-BAR结构域的突触后蛋白，在果蝇中调节肌肉横管（T管）的形成。我们通过免疫共沉淀验证了这种相互作用，并显示dAmph-SH3结构域与dNedd4Lo N端之间存在直接结合。因此，dNedd4Lo与dAmph在突触后和肌肉T管处共定位。此外，在胚胎发育期间在肌肉中表达dNedd4Lo导致dAmph消失和T管形成受损，模拟了amph基因敲除突变体的表型。在表达dNedd4S或催化失活的dNedd4Lo（C→A）的肌肉中未观察到这种效应。我们提出，dNedd4Lo使肌肉中的dAmph不稳定，导致T管形成受损和肌肉功能受损。
