McGough Ian J, Steinberg Florian, Jia Da, Barbuti Peter A, McMillan Kirsty J, Heesom Kate J, Whone Alan L, Caldwell Maeve A, Billadeau Daniel D, Rosen Michael K, Cullen Peter J
The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Curr Biol. 2014 Jul 21;24(14):1670-1676. doi: 10.1016/j.cub.2014.06.024. Epub 2014 Jul 3.
Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease [2, 3]. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.
逆转录复合物是一种蛋白质组装体，在协调跨膜货物蛋白从内体输出到前往高尔基体和质膜的回收途径中发挥核心作用[1]。最近，逆转录复合物成分VPS35的一种特定突变，即VPS35(D620N)，已将逆转录复合物功能障碍与家族性常染色体显性和散发性帕金森病联系起来[2,3]。然而，这种突变对逆转录复合物功能的影响仍不清楚。在这里，我们证实，在表达VPS35(D620N)的细胞中，内体到反式高尔基体网络（TGN）的运输受到干扰，但内体到质膜的再循环不受影响，我们在携带VPS35(D620N)突变的患者细胞中也证实了这一点。通过基于细胞培养中氨基酸的比较稳定同位素标记（SILAC）分析野生型VPS35与VPS35(D620N)突变体的相互作用组，我们确定D620N突变的主要缺陷在于与肌动蛋白成核的威斯科特-奥尔德里奇综合征和SCAR同源物（WASH）复合物的结合。此外，使用等温滴定量热法，我们确定VPS35(D620N)突变体的主要缺陷是对WASH复合物成分FAM21的亲和力降低了2.2±0.5倍。这些数据定义了由VPS35(D620N)突变引起的逆转录复合物组装中的主要分子缺陷，并通过揭示对逆转录复合物介导的内体到TGN运输的功能影响，为帕金森病中逆转录复合物失调提供了新的见解。
