Mammalian arrestins have a major role in the intracellular trafficking of seven-transmembrane (7TM) receptors. The fungal ambient pH signaling pathway involves an arrestin-related protein, PalF/Rim8, and the ESCRT (endosomal sorting complex required for transport) machinery. We found that in Saccharomyces cerevisiae, Rim8 binds to both the putative 7TM pH sensor Rim21 and the ESCRT-I subunit Vps23. We show that an SXP motif in Rim8 mediates binding to the Vps23 ubiquitin E2 variant (UEV) domain and that a monoubiquitinated residue near the SXP motif contributes to this interaction. We present evidence that Rim8 ubiquitination is dependent on the Rsp5 E3 ubiquitin ligase and triggered upon binding of Vps23 UEV to both the SXP motif and ubiquitin, thus suggesting a two-step binding mechanism. We further show that Rim8 coimmunoprecipitates with ESCRT-I subunits Vps23 and Vps28, supporting the idea that binding of Rim8 to Vps23 mediates the association of Rim8 with the ESCRT-I complex. Fluorescence microscopic analyses indicate that overexpressed Rim8 and Vps23 colocalize at cortical punctate structures, providing additional evidence of the interaction between these two proteins. Strikingly, our findings indicate that evolutionary conserved mechanisms control the recruitment of the ESCRT machinery to Pal/Rim proteins in fungi and retroviral Gag proteins in animal cells.Arrestins play an essential role in the regulation of seventransmembrane (7TM) receptors (31). Although initially identified on the basis of their ability to uncouple 7TM receptors from heterotrimeric G proteins in a process known as desensitization, arrestins were later found to serve as endocytic adaptors that recruit clathrin and the clathrin adaptor protein AP-2 and facilitate 7TM receptor internalization via clathrincoated vesicles (17,29,30).Until recently, arrestins were thought to be restricted to the animal kingdom. However, the identification of an arrestin-related protein in Aspergillus nidulans, PalF, demonstrated that members of this protein family are also present in fungi (22). PalF and its yeast homolog Rim8 are involved in the Pal/Rim signaling pathway, which mediates the ambient pH response and is activated in neutral-alkaline environments (41). As mammalian beta-arrestins, PalF, which contains arrestin N-terminal and C-terminal domains, binds to the cytoplasmic domain of a 7TM protein, the putative pH sensor PalH, and is ubiquitinated in a signal-and 7TM receptor-dependent manner (22).The PalF/Rim8 signaling function does not appear to be related to receptor desensitization, as there is no evidence of G protein involvement in the Pal/Rim pathway. However, evidence strongly suggests that these proteins, as mammalian ␤-arrestins, play a role as endocytic adaptors. Most of the components of the ESCRT (endosomal sorting complex required for transport) machinery involved in the MVB (multivesicular body) sorting pathway (24, 48), play an essential role in the Pal/Rim signaling pathway (4,7,12,15,20,28,43,45,62). According to the current m...

The pathogenic Old World arenavirus Lassa virus (LASV) causes a severe hemorrhagic fever with a high rate of mortality in humans. Several LASV receptors, including dystroglycan (DG), TAM receptor tyrosine kinases, and C-type lectins, have been identified, suggesting complex receptor use. Upon receptor binding, LASV enters the host cell via an unknown clathrin-and dynamin-independent pathway that delivers the virus to late endosomes, where fusion occurs. Here we investigated the mechanisms underlying LASV endocytosis in human cells in the context of productive arenavirus infection, using recombinant lymphocytic choriomeningitis virus (rLCMV) expressing the LASV glycoprotein (rLCMV-LASVGP). We found that rLCMV-LASVGP entered human epithelial cells via DG using a macropinocytosis-related pathway independently of alternative receptors. Dystroglycan-mediated entry of rLCMV-LASVGP required sodium hydrogen exchangers, actin, and the GTPase Cdc42 and its downstream targets, p21-activating kinase-1 (PAK1) and Wiskott-Aldrich syndrome protein (N-Wasp). Unlike other viruses that enter cells via macropinocytosis, rLCMV-LASVGP entry did not induce overt changes in cellular morphology and hardly affected actin dynamics or fluid uptake. Screening of kinase inhibitors identified protein kinase C, phosphoinositide 3-kinase, and the receptor tyrosine kinase human hepatocyte growth factor receptor (HGFR) to be regulators of rLCMV-LASVGP entry. The HGFR inhibitor EMD 1214063, a candidate anticancer drug, showed antiviral activity against rLCMV-LASVGP at the level of entry. When combined with ribavirin, which is currently used to treat human arenavirus infection, EMD 1214063 showed additive antiviral effects. In sum, our study reveals that DG can link LASV to an unusual pathway of macropinocytosis that causes only minimal perturbation of the host cell and identifies cellular kinases to be possible novel targets for therapeutic intervention. IMPORTANCELassa virus (LASV) causes several hundred thousand infections per year in Western Africa, with the mortality rate among hospitalized patients being high. The current lack of a vaccine and the limited therapeutic options at hand make the development of new drugs against LASV a high priority. In the present study, we uncover that LASV entry into human cells via its major receptor, dystroglycan, involves an unusual pathway of macropinocytosis and define a set of cellular factors implicated in the regulation of LASV entry. A screen of kinase inhibitors revealed HGFR to be a possible candidate target for antiviral drugs against LASV. An HGFR candidate inhibitor currently being evaluated for cancer treatment showed potent antiviral activity and additive drug effects with ribavirin, which is used in the clinic to treat human LASV infection. In sum, our study reveals novel fundamental aspects of the LASV-host cell interaction and highlights a possible candidate drug target for therapeutic intervention. The Old World arenavirus Lassa virus (LASV) is the causative agent of a severe viral ...

In metazoans, proteins of the arrestin family are key players of G-protein-coupled receptors (GPCRS) signaling and trafficking. Following stimulation, activated receptors are phosphorylated, thus allowing the binding of arrestins and hence an “arrest” of receptor signaling. Arrestins act by uncoupling receptors from G proteins and contribute to the recruitment of endocytic proteins, such as clathrin, to direct receptor trafficking into the endocytic pathway. Arrestins also serve as adaptor proteins by promoting the recruitment of ubiquitin ligases and participate in the agonist-induced ubiquitylation of receptors, known to have impact on their subcellular localization and stability. Recently, the arrestin family has expanded following the discovery of arrestin-related proteins in other eukaryotes such as yeasts or fungi. Surprisingly, most of these proteins are also involved in the ubiquitylation and endocytosis of plasma membrane proteins, thus suggesting that the role of arrestins as ubiquitin ligase adaptors is at the core of these proteins' functions. Importantly, arrestins are themselves ubiquitylated, and this modification is crucial for their function. In this paper, we discuss recent data on the intricate connections between arrestins and the ubiquitin pathway in the control of endocytosis.

The yeast Rim8/Art9 α-arrestin, involved in ambient pH signaling, is regulated through multisite phosphorylation of the hinge region by the plasma membrane–associated casein kinase 1. This modification prevents its stable association with the pH sensor protein Rim21 at the plasma membrane and thereby inhibits signal transduction at acidic pH.

Background:The mechanisms by which ubiquitin chain length is regulated remain largely unknown. Results: A ubiquitin-binding domain (UBD) acts in trans to direct substrate monoubiquitination in vitro. Conclusion: UBD binding to the first conjugated ubiquitin prevents further polyubiquitination. Significance: We report a new mechanism to control ubiquitin chain elongation.

Recognition of functional receptors by viruses is a key determinant for their host range, tissue tropism, and disease potential. The highly pathogenic Lassa virus (LASV) currently represents one of the most important emerging pathogens. The major cellular receptor for LASV in human cells is the ubiquitously expressed and evolutionary highly conserved extracellular matrix receptor dystroglycan (DG). In the host, DG interacts with many cellular proteins in a tissue-specific manner. The resulting distinct supramolecular complexes likely represent the functional units for viral entry, and preexisting protein-protein interactions may critically influence DG’s function in productive viral entry. Using an unbiased shotgun proteomic approach, we define the largely unknown molecular composition of DG complexes present in highly susceptible epithelial cells that represent important targets for LASV during viral transmission. We further show that the specific composition of cellular DG complexes can affect DG’s function in receptor-mediated endocytosis of the virus. Under steady-state conditions, epithelial DG complexes underwent rapid turnover via an endocytic pathway that shared some characteristics with DG-mediated LASV entry. However, compared to steady-state uptake of DG, LASV entry via DG occurred faster and critically depended on additional signaling by receptor tyrosine kinases and the downstream effector p21-activating kinase. In sum, we show that the specific molecular composition of DG complexes in susceptible cells is a determinant for productive virus entry and that the pathogen can manipulate the existing DG-linked endocytic pathway. This highlights another level of complexity of virus-receptor interaction and provides possible cellular targets for therapeutic antiviral intervention. IMPORTANCE Recognition of cellular receptors allows emerging viruses to break species barriers and is an important determinant for their disease potential. Many virus receptors have complex tissue-specific interactomes, and preexisting protein-protein interactions may influence their function. Combining shotgun proteomics with a biochemical approach, we characterize the molecular composition of the functional receptor complexes used by the highly pathogenic Lassa virus (LASV) to invade susceptible human cells. We show that the specific composition of the receptor complexes affects productive entry of the virus, providing proof-of-concept. In uninfected cells, these functional receptor complexes undergo dynamic turnover involving an endocytic pathway that shares some characteristics with viral entry. However, steady-state receptor uptake and virus endocytosis critically differ in kinetics and underlying signaling, indicating that the pathogen can manipulate the receptor complex according to its needs. Our study highlights a remarkable complexity of LASV-receptor interaction and identifies possible targets for therapeutic antiviral intervention.