背景:将货物从内体运输到跨高尔基体网络需要许多顺序和协调的步骤。货物被分类为内体来源的载体,这些载体被运输,系留,融合到跨高尔基网络上.系链步骤需要几个复合物,包括高尔基相关的逆行蛋白复合物,其在跨高尔基体网络上的定位由Arl和Rab家族的小GTP酶的活性决定。然而,高尔基体相关的逆行蛋白复合物如何识别内体衍生的载体,这些载体将与跨高尔基体网络融合,目前尚不清楚.
方法:我们通过在过表达Rab4b的细胞中使用荧光货物或在小干扰RNA敲低Rab4b后,结合高尔基体相关逆行蛋白复合物亚基的下调,研究了向反式高尔基体网络的逆行运输。我们使用免疫荧光和图像处理(超分辨率径向波动和3D重建)以及生化方法来表征这些干预措施对货运公司贩运的后果。
结果:我们报道了高尔基体相关逆行蛋白复合物的VPS52亚基是Rab4b的效应子。我们发现野生型或活性Rab4b的过表达以高尔基体相关的逆行蛋白复合物依赖性方式增加了阳离子非依赖性甘露糖-6-磷酸受体的早期内体到反式高尔基体网络的逆行运输。相反,无活性Rab4b或Rab4b敲低的过表达减弱了这种贩运。在没有Rab4b的情况下,内化的不依赖阳离子的甘露糖6磷酸受体无法获得看起来像内体亚结构域和/或内体衍生载体的VPS52标记的结构,其亚细胞分布与Rab4b无关。因此,非阳离子依赖性甘露糖-6-磷酸受体在早期内体中被阻断,并且不再能够进入反式高尔基体网络.
结论:我们的结果支持Rab4b,通过控制阳离子非依赖性甘露糖-6-磷酸受体向VPS52微域的分选,赋予货物承运人前往跨高尔基网络的方向特异性。鉴于胞吞再循环在细胞稳态中的重要性,Rab4b/高尔基体相关的逆行蛋白复合物依赖性步骤的破坏可能对病理产生严重后果.
BACKGROUND: The trafficking of cargoes from endosomes to the trans-Golgi network requires numerous sequential and coordinated steps. Cargoes are sorted into endosomal-derived carriers that are transported, tethered, and fused to the trans-Golgi network. The tethering step requires several complexes, including the Golgi-associated retrograde protein complex, whose localization at the trans-Golgi network is determined by the activity of small GTPases of the Arl and Rab family. However, how the Golgi-associated retrograde protein complex recognizes the endosome-derived carriers that will fuse with the trans-Golgi network is still unknown.
METHODS: We studied the retrograde trafficking to the trans-Golgi network by using fluorescent cargoes in cells overexpressing Rab4b or after Rab4b knocked-down by small interfering RNA in combination with the downregulation of subunits of the Golgi-associated retrograde protein complex. We used immunofluorescence and image processing (Super Resolution Radial Fluctuation and 3D reconstruction) as well as biochemical approaches to characterize the consequences of these interventions on cargo carriers trafficking.
RESULTS: We reported that the VPS52 subunit of the Golgi-associated retrograde protein complex is an effector of Rab4b. We found that overexpression of wild type or active Rab4b increased early endosomal to trans-Golgi network retrograde trafficking of the cation-independent mannose-6-phosphate receptor in a Golgi-associated retrograde protein complex-dependent manner. Conversely, overexpression of an inactive Rab4b or Rab4b knockdown attenuated this trafficking. In the absence of Rab4b, the internalized cation-independent mannose 6 phosphate receptor did not have access to VPS52-labeled structures that look like endosomal subdomains and/or endosome-derived carriers, and whose subcellular distribution is Rab4b-independent. Consequently, the cation-independent mannose-6-phosphate receptor was blocked in early endosomes and no longer had access to the trans-Golgi network.
CONCLUSIONS: Our results support that Rab4b, by controlling the sorting of the cation-independent mannose-6-phosphate receptor towards VPS52 microdomains, confers a directional specificity for cargo carriers en route to the trans-Golgi network. Given the importance of the endocytic recycling in cell homeostasis, disruption of the Rab4b/Golgi-associated retrograde protein complex-dependent step could have serious consequences in pathologies.