SNX25 regulates TGF-β signaling by enhancing the receptor degradation

SNXs (sorting nexin), a family of proteins playing roles in cargo sorting and signaling from compartments within the endocytic network, regulate traffic of membrane proteins including TGF-β receptors. Here we report that the full length human and mouse SNX25, a SNX member with PX, PXA and RGS domains, co-localizes with TGF-β receptors, and forms internalized cytosolic punctae upon treatment with TGF-β. While overexpression of SNX25 inhibits TGF-β induced luciferase reporter activity, knocking down endogenous SNX25 by siRNA in NIH3T3 cells elevates the TGF-β receptor levels and facilitates TGF-β signaling. Immunoprecipitation experiments demonstrate that SNX25 interacts with TβRI. Western blot analyses indicate that SNX25 enhances the degradation of TGF-β receptors. SNX25 induced TGF-β receptor degradation is shown via the clathrin dependent endocytosis pathway into lysosome. We have characterized that PXA domain of SNX25 is required for the degradation of TβRI. Our findings demonstrate that SNX25 negatively regulates TGF-β signaling by enhancing the receptor degradation through lysosome pathway.     

Structure of Sorting Nexin 11 (SNX11) Reveals a Novel Extended Phox Homology (PX) Domain Critical for Inhibition of SNX10-induced Vacuolation

Sorting nexins are phox homology (PX) domain-containing proteins involved in diverse intracellular endosomal trafficking pathways. The PX domain binds to certain phosphatidylinositols and is recruited to vesicles rich in these lipids. The structure of the PX domain is highly conserved, containing a three-stranded β-sheet, followed by three α-helices. Here, we report the crystal structures of truncated human SNX11 (sorting nexin 11). The structures reveal that SNX11 contains a novel PX domain, hereby named the extended PX (PXe) domain, with two additional α-helices at the C terminus. We demonstrate that these α-helices are indispensible for the in vitro functions of SNX11. We propose that this PXe domain is present in SNX10 and is responsible for the vacuolation activity of SNX10. Thus, this novel PXe domain constitutes a structurally and functionally important PX domain subfamily.     

重组人源SNX7蛋白在大肠杆菌中的表达、纯化及磷脂结合特异性分析

Sorting nexins(SNXs)是一类含有SNX-PX结构域,并在细胞内吞和内体分选运输过程中发挥重要调节作用的蛋白。SNX7是SNXs家族中的一员,含有PX结构域和BAR结构域,属于SNX-PX-BAR亚家族。斑马鱼实验表明,SNX7是在肝脏中大量表达的抗凋亡蛋白,并在胚胎肝脏的发育中发挥关键作用。为了从蛋白水平对SNX7进行研究,首先将编码人源PX-BARSNX7(SNX7的一个片段,包含PX和BAR结构域)的cDNA片段插入到原核表达载体p28a中,再将重组质粒转化到大肠杆菌Rosseta 2(DE3)中诱导表达,并用亲和层析、离子交换和分子筛层析对PX-BARSNX7进行了纯化。Western blotting结果表明,亲和层析、离子交换和分子筛层析纯化后获得了高纯度的PX-BARSNX7蛋白。动态光散射实验显示PX-BARSNX7蛋白均一性良好。磷脂结合实验表明,PX-BARSNX7具有较为广泛的磷脂酰肌醇结合能力,能够与PtdIns(5)P、PtdIns(4,5)P2和PtdIns(3,4,5)P3结合。     

Inhibitory regulation of EGF receptor degradation by sorting nexin 5

Endosomal trafficking of EGF receptor (EGFR) upon stimulation is a highly regulated process during receptor-mediated signaling. Recently, the sorting nexin (SNX) family has emerged as an important regulator in the membrane trafficking of EGFR. Here, we report the identification of a novel interaction between two members of the family, SNX1 and SNX5, which is mediated by the newly defined BAR domain of both SNXs. We have also shown that the PX domain of SNX5 binds specifically to PtdIns other than to PtdIns(3)P. Furthermore, the BAR domain but not the PX domain of SNX5 is sufficient for its subcellular membrane association. Functionally, overexpression of SNX5 inhibits the degradation of EGFR. This process appears to be independent of its interaction with SNX1. However, overexpression of SNX1 is able to attenuate the effect of SNX5 on EGFR degradation, suggesting the two proteins may play antagonistic roles in regulating endosomal trafficking of the receptor.     

Expression of Sorting Nexin 18 (SNX18) Is Dynamically Regulated in Developing Spinal Motor Neurons

The sorting nexin (SNX) family proteins, which contain a Phox homology (PX) domain, play crucial roles in regulating the intracellular membrane trafficking of the endocytic pathway. The proper coordination of this pathway is important for axonal elongation; however, little is known about the expression and intracellular dynamics of the SNX members during the formation of the nervous system. Here the authors found that SNX18, which belongs to the Src-homology-3-PX-Bin/Amphiphysin/Rvs domain-containing SNX subfamily, was specifically expressed in differentiating motor neurons in the chick and mouse embryonic spinal cord. The expression of SNX18 in embryonic spinal motor neurons was transient and was downregulated as the neurons matured. The authors further demonstrated that the localization of EGFP-SNX18 in growth cones was dynamically regulated and accumulated especially at areas in contact with permissive substrates. These findings collectively suggest that SNX18 may play an active role in axonal elongation.     

AP-1 and retromer play opposite roles in the trafficking of sortilin between the Golgi apparatus and the lysosomes

Sortilin has been implicated in the sorting of one soluble hydrolase and two sphingolipid activator proteins to the lysosomes. While the GGA adaptor proteins have been demonstrated to play a role in the targeting of sortilin to the endosomes, the recycling of sortilin has not yet been elucidated. Here we examine the role of two adaptor protein complexes, AP-1 and retromer. Our results demonstrate that AP-1 is required for the transport of sortilin to the endosomes and retromer for the recycling of sortilin to the Golgi apparatus. While inhibition of AP-1 causes accumulation of sortilin in the Golgi apparatus, RNAi depletion of retromer results in retention of sortilin in the lysosomes. We also demonstrate that the interaction of sortilin with retromer occurs through a YXXΦ site in its cytosolic tail. In conclusion, our observations indicate that retromer and AP-1 play opposite roles in the trafficking of sortilin.     

Expression of a novel member of sorting nexin gene family,SNX-L,in human liver development

The sorting nexin (SNX) protein family is implicated in the regulation of receptor degradation and membrane traffic in the cell. With the aim of identifying novel genes involved in receptor degradation and recycling, we have cloned a new member of the sorting nexin gene family, human sorting nexin L, SNX-L (or SNX21). This gene includes 4 exons and 3 introns, and is located on chromosome 20q12-13.1 region, encompassing 8 kb. The full-length cDNA of SNX-L is 1,811 bp, with an open reading frame of 1,092 bp. The protein consists of 364 amino acids and encodes a 40 kDa protein. The SNX-L protein has a common PX domain shared by all SNX family members. The similarity of SNX-L PX domain to the PX consensus sequence is over 40%. PX domains have been shown to associate with specific phospholipids and membrane compartments. Expression analysis of SNX-L mRNA indicates that SNX-L is distinctly and highly expressed in fetus liver, but only weakly expressed in brain, muscle (skeleton muscle, smooth muscle, and cardiac muscle), kidney, and adrenal gland. Strong liver expression of SNX-L is maintained from 12 to 25 weeks during human fetus development, suggesting that SNX-L may be a regulatory gene involved in receptor protein degradation during embryonic liver development.     

Clathrin and AP1B: Key roles in basolateral trafficking through trans-endosomal routes

Research following introduction of the MDCK model system to study epithelial polarity (1978) led to an initial paradigm that posited independent roles of the trans Golgi network (TGN) and recycling endosomes (RE) in the generation of, respectively, biosynthetic and recycling routes of plasma membrane (PM) proteins to apical and basolateral PM domains. This model dominated the field for 20 years. However, studies over the past decade and the discovery of the involvement of clathrin and clathrin adaptors in protein trafficking to the basolateral PM has led to a new paradigm. TGN and RE are now believed to cooperate closely in both biosynthetic and recycling trafficking routes. Here, we critically review these recent advances and the questions that remain unanswered.     

Sorting nexin 8 regulates endosome-to-Golgi transport

Sorting nexin 8 (SNX8) belongs to the sorting nexin protein family, whose members are involved in endocytosis and endosomal sorting and signaling. The function of SNX8 has so far been unknown. Here, we have investigated the role of SNX8 in intracellular transport of the bacterial toxin Shiga toxin (Stx) and the plant toxin ricin. After being endocytosed, these toxins are transported retrogradely from endosomes, via the Golgi apparatus and the endoplasmic reticulum (ER), into the cytosol, where they exert their toxic effect. Interestingly, our experiments show that SNX8 regulates the transport of Stx and ricin differently; siRNA-mediated knockdown of SNX8 significantly increased the Stx transport to the trans-Golgi network (TGN), whereas ricin transport was slightly inhibited. We also found that SNX8 colocalizes with early endosome antigen 1 (EEA1) and with retromer components, suggesting an endosomal localization of SNX8 and supporting our finding that SNX8 is involved in endosomal sorting.