The redox sensor TXNL1 plays a regulatory role in fluid phase endocytosis.

BACKGROUND: Small GTPases of the Rab family can cycle between a GTP- and a GDP-bound state and also between membrane and cytosol. The latter cycle is mediated by the Guanine Nucleotide Dissociation Inhibitor GDI, which can selectively extract GDP-bound Rab proteins from donor membranes, and then reload them on target membranes. In previous studies, we found that capture of the small GTPase Rab5, a key regulator of endocytic membrane traffic, by GDI is stimulated by oxidative stress via p38MAPK, resulting in increased fluid phase endocytosis. METHODOLOGY/PRINCIPAL FINDINGS: When purifying the GDI stimulating activity we found that that it copurified with a high MW protein complex, which included p38MAPK. Here we report the identification and characterization of another component of this complex as the thioredoxin-like protein TXNL1. Our observations indicate that TXNL1 play a selective role in the regulation of fluid phase endocytosis, by controlling GDI capacity to capture Rab5. CONCLUSIONS/SIGNIFICANCE: Oxidants, which are known to cause cellular damage, can also trigger signaling pathways, in particular via members of the thioredoxin family. We propose that TXNL1 acts as an effector of oxidants or a redox sensor by converting redox changes into changes of GDI capacity to capture Rab5, which in turn modulates fluid phase endocytosis.

Intracellular trafficking of interleukin-1 receptor I requires Tollip.

Interleukin-1 receptor (IL-1RI) is a master regulator of inflammation and innate immunity. When triggered by IL-1beta, IL-1RI aggregates with IL-1R-associated protein (IL-1RAcP) and forms a membrane proximal signalosome that potently activates downstream signaling cascades. IL-1beta also rapidly triggers endocytosis of IL-1RI. Although internalization of IL-1RI significantly impacts signaling, very little is known about trafficking of IL-1RI and therefore about precisely how endocytosis modulates the overall cellular response to IL-1beta. Upon internalization, activated receptors are often sorted through endosomes and delivered to lysosomes for degradation. This is a highly regulated process that requires ubiquitination of cargo proteins as well as protein-sorting complexes that specifically recognize ubiquitinated cargo. Here, we show that IL-1beta induces ubiquitination of IL-1RI and that via these attached ubiquitin groups, IL-1RI interacts with the ubiquitin-binding protein Tollip. By using an assay to follow trafficking of IL-1RI from the cell surface to late endosomes and lysosomes, we demonstrate that Tollip is required for sorting of IL-1RI at late endosomes. In Tollip-deficient cells and cells expressing only mutated Tollip (incapable of binding IL-1RI and ubiquitin), IL-1RI accumulates on late endosomes and is not efficiently degraded. Furthermore, we show that IL-1RI interacts with Tom1, an ubiquitin-, clathrin-, and Tollip-binding protein, and that Tom1 knockdown also results in the accumulation of IL-1RI at late endosomes. Our findings suggest that Tollip functions as an endosomal adaptor linking IL-1RI, via Tom1, to the endosomal degradation machinery.

Capture of the small GTPase Rab5 by GDI: regulation by p38 MAP kinase.

The small GTPase Rab5 is one of the key regulators of early endocytic traffic and, like other GTPases, cycles between GTP- and GDP-bound states as well as between membrane and cytosol. The latter cycle is controlled by a guanine nucleotide dissociation inhibitor (GDI), which functions as a Rab vehicle in the cytosol. GDI extracts from membranes the inactive GDP-bound form of the Rab. Then, the cytosolic GDI:Rab complex is delivered to the appropriate target membrane, where the Rab protein is reloaded, presumably via a GDI displacement factor (Pfeffer and Aivazian, 2004). We previously reported that the formation of the GDI:Rab5 complex is stimulated by the mitogen-activated protein kinase p38 (Cavalli et al., 2001). Mol. Cell7, 421-432.]. Selective activation of p38 MAPK increases endocytic rates in vivo, presumably allowing more efficient internalization of cell surface components for repair, storage, or degradation. These observations emphasize the possibility that external stimuli contribute to the regulation of membrane traffic. Here, we describe how to monitor the ability of GDI to extract Rab5 from early endosomal membranes in vitro and the role of p38 MAPK in this process. In addition, we detail how to investigate the possible role of p38 MAPK in the regulation of endocytosis in vivo.

Signaling from the far side.

Signaling by cell surface receptors is often turned off by receptor endocytosis and downregulation. However, it appears that some signaling pathways continue to fire from within cells. A recent study now suggests that a late endosomal p14/MP1-MAPK scaffold complex is critical for the ERK signaling pathway.