Distinct complexes of yeast Snx4 family SNX‐BARs mediate retrograde trafficking of Snc1 and Atg27

The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4‐Atg20 and Snx4‐Snx41. Each heterodimer coats an endosome‐derived tubule that mediates retrograde sorting of distinct cargo; the v‐SNARE, Snc1, is a cargo of the Snx4‐Atg20 pathway, and Snx4‐Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5‐Vps17 retromer SNX‐BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer‐coated tubules, promotes fission of Snx4‐Atg20 coated tubules. The results indicate that the yeast SNX‐BAR proteins coat 3 distinct types of endosome‐derived carriers that mediate endosome‐to‐Golgi retrograde trafficking.      

Sorting nexin 3, a protein upregulated by lithium, contains a novel phosphatidylinositol-binding sequence and mediates neurite outgrowth in N1E-115 cells

Lithium, a drug in the treatment of bipolar disorder, modulates many aspects of neuronal developmental processes such as neurogenesis, survival, and neuritogenesis. However, the underlying mechanism still remains to be understood. Here, we show that lithium upregulates the expression of sorting nexin 3 (SNX3), one of the Phox (PX) domain-containing proteins involved in endosomal sorting, and regulates neurite outgrowth in mouse N1E-115 neuroblastoma cells. The inhibition of SNX3 function by its knockdown decreases lithium-induced outgrowth of neurites. Transfection of the full-length SNX3 construct into cells facilitates the outgrowth. We also find that the C-terminus, as well as the PX domain, of SNX3 has a functional binding sequence with phosphatidylinositol monophosphates. Transfection of the C-terminal deletion mutant or only the C-terminus does not have an effect on the outgrowth. These results suggest that SNX3, a protein upregulated by lithium, is an as yet unknown regulator of neurite formation and that it contains another functional phosphatidylinositol phosphate-binding region at the C-terminus.     

Structure of human SNX10 reveals insights into its role in human autosomal recessive osteopetrosis

Sorting nexin 10 (SNX10), the unique member of the SNX family having vacuolation activity in cells, was shown to be involved in the development of autosomal recessive osteopetrosis (ARO) in recent genetic studies. However, the molecular mechanism of the disease‐related mutations affecting the biological function of SNX10 is unclear. Here, we report the crystal structure of human SNX10 to 2.6Å resolution. The structure reveals that SNX10 contains the extended phox‐homology domain we previously proposed. Our study provides the structural details of those disease‐related mutations. Combined with the vacuolation study of those mutations, we found that Tyr32 and Arg51 are important for the protein stability and both play a critical role in vacuolation activity, while Arg16Leu may affect the function of SNX10 in osteoclast through protein–protein interactions. Proteins 2014; 82:3483–3489. © 2014 Wiley Periodicals, Inc.     

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.     

Inhibition of Na,K-ATPase-suppressive activity of translationally controlled tumor protein by sorting nexin 6

Translationally controlled tumor protein (TCTP) has both extra- and intracellular functions. Our group recently reported that TCTP interacts with Na,K-ATPase and suppresses its activity. Our studies led to the identification of sorting nexin 6 (SNX6) which binds with TCTP as a potential negative regulator of TCTP. SNX6 does not interact directly with any cytoplasmic domains of Na,K-ATPase. However, when overexpressed, it restores the Na,K-ATPase activity suppressed by TCTP. This was confirmed by measurements of purified plasma membrane Na,K-ATPase activity after incubation with recombinant TCTP and SNX6. SNX6 alone has no effect on Na,K-ATPase activity, but activates Na,K-ATPase via inhibition of TCTP. Inhibition of endogenous TCTP by the overexpression of SNX6 or knockdown of TCTP expression by siTCTP increased Na,K-ATPase activity above the basal level. The interaction between SNX6 and TCTP thus appears to regulate Na,K-ATPase activity.     

SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless

Drosophila Wingless (Wg) acts as a morphogen during development. Wg secretion is controlled by a seven-pass transmembrane cargo Wntless (Wls). We have recently identified retromer as a key regulator involved in Wls trafficking. As sorting nexin (SNX) molecules are essential components of the retromer complex, we hypothesized that specific SNX(s) is required for retromer-mediated Wnt secretion. Here, we generated Drosophila mutants for all of the eight snx members, and identified Drosophila SNX3 (DSNX3) as an essential molecule required for Wg secretion. We show that Wg secretion and its signaling activity are defective in Dsnx3 mutant clones in wing discs. Wg levels in the culture medium of Dsnx3-depleted S2 cells are also markedly reduced. Importantly, Wls levels are strikingly reduced in Dsnx3 mutant cells, and overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. Moreover, DSNX3 can interact with the retromer component Vps35, and co-localize with Vps35 in early endosomes. These data indicate that DSNX3 regulates Wg secretion via retromer-dependent Wls recycling. In contrast, we found that Wg secretion is not defective in cells mutant for Drosophila snx1 and snx6, two components of the classical retromer complex. Ectopic expression of DSNX1 or DSNX6 fails to rescue the Wg secretion defect in Dsnx3 mutant wing discs and in Dsnx3 dsRNA-treated S2 cells. These data demonstrate the specificity of the DSNX3-retromer complex in Wls recycling. Together, our findings suggest that DSNX3 acts as a cargo-specific component of retromer, which is required for endocytic recycling of Wls and Wg/Wnt secretion.     

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.     

Diacylglycerol kinase ζ: at the crossroads of lipid signaling and protein complex organization

Diacylglycerol (DAG) and phosphatidic acid (PA) are lipids with unique functions as metabolic intermediates, basic membrane constituents, and second-signal components. Diacylglycerol kinases (DGK) regulate the levels of these two lipids, catalyzing the interconversion of one to the other. The DGK family of enzymes is composed of 10 isoforms, grouped into five subfamilies based on the presence of distinct regulatory domains. From its initial characterization as a type IV DGK to the generation of mouse models showing its importance in cardiac dysfunction and immune pathologies, diacylglycerol kinase ζ (DGKζ) has proved an excellent example of the critical role of lipid-metabolizing enzymes in the control of cell responses. Although the mechanism that regulates this enzyme is not well known, many studies demonstrate its subtle regulation and its strategic function in specific signaling and as part of adaptor protein complexes. These data suggest that DGKζ offers new opportunities for therapeutic manipulation of lipid metabolism.     

An unexpected role for PI4,5P2 in EGF receptor endosomal trafficking

In mammalian cells, three Cdc25 phosphatases A, B, C coordinate cell cycle progression through activating dephosphorylation of Cyclin-dependent kinases. Whereas Cdc25B is believed to trigger entry into mitosis, Cdc25C is thought to act at a later stage of mitosis and in the nucleus. We report that a fraction of Cdc25C localises to centrosomes in a cell cycle-dependent fashion, as of late S phase and throughout G2 and mitosis. Moreover, Cdc25C colocalises with Cyclin B1 at centrosomes in G2 and in prophase and Fluorescence Recovery after Photobleaching experiments reveal that they are both in dynamic exchange between the centrosome and the cytoplasm. The centrosomal localisation of Cdc25C is essentially mediated by its catalytic C-terminal domain, but does not require catalytic activity. In fact phosphatase-dead and substrate-binding hotspot mutants of Cdc25C accumulate at centrosomes together with phosphoTyr15-Cdk1 and behave as dominant negative forms that impair entry into mitosis. Taken together, our data suggest an unexpected function for Cdc25C at the G2/M transition, in dephosphorylation of Cdk1. We propose that Cdc25C may participate in amplification of Cdk1-Cyclin B1 activity following initial activation by Cdc25B, and that this process is initiated at the centrosome, then further propagated throughout the cytoplasm thanks to the dynamic behavior of both Cdc25C and Cyclin B1.     

Sorting nexin 17, a non-self-assembling and a PtdIns(3)P high class affinity protein, interacts with the cerebral cavernous malformation related protein KRIT1

The mammalian sorting nexin (SNX) proteins are involved in the endocytosis and the sorting machinery of transmembrane proteins. Additionally to the family defining phox homology (PX) domain, SNX17 is the only member with a truncated FERM (4.1, ezrin, radixin, and moesin) domain and a unique C-terminal region (together designated as FC unit). By gel filtration and lipid overlay assays we show that SNX17 is a non-self-assembling and a PtdIns(3)P high class affinity protein. A SNX17 affinity to any other phosphoinositides was not detected. By yeast two-hybrid- and GST-trapping assays we identified KRIT1 (krev1 interaction trapped 1) as a new specific interaction partner of the FC unit of SNX17. KRIT1 binds SNX17 by its N-terminal region like the known interaction partner ICAP1alpha (integrin cytoplasmic domain-associated protein-1). The interaction was also detected in HEK 293 cells transiently expressing GFP-tagged KRIT1 and Xpress-tagged SNX17. KRIT1 mutations cause cerebral cavernous malformation (CCM1). Our finding suggests a SNX17 involvement in the indicated KRIT1 function in cell adhesion processes by integrin signaling.