Regulation of WASH-Dependent Actin Polymerization and Protein Trafficking by Ubiquitination

Department of Physiology, UT Southwestern Dallas, TX 75390, USA.
Cell (Impact Factor: 32.24). 02/2013; 152(5):1051-64. DOI: 10.1016/j.cell.2013.01.051
Source: PubMed


Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.

Download full-text


Available from: Yi-Heng Hao, Sep 02, 2015
46 Reads
  • Source
    • "UBE2C N-terminal extension plays a role in the ubiquitination mechanism both at the ubiquitination and target lysine levels (Summers et al., 2008) while UBE2R2 C-terminal extension may be implicated in both ubiquitination and localization (Sadowski et al., 2007). The large UBE2O is per se a combined E2-E3 entity (Berleth and Pickart, 1996) but it is also able to work with the E3 MAGE-L2/TRIM27, suggesting multi-functional roles for this E2 (Hao et al., 2013). More than half of the E2 enzymes exhibit extensions and future studies will have to clearly identify their roles, which may encompass localization, recognition of substrates, binding with specific E3s, modulation of activity, etc. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Ubiquitin Proteasome System (UPS) is a major actor of muscle wasting during various physio-pathological situations. In the past 15 years, increasing amounts of data have depicted a picture, although incomplete, of the mechanisms implicated in myofibrillar protein degradation, from the discovery of muscle-specific E3 ligases to the identification of the signaling pathways involved. The targeting specificity of the UPS relies on the capacity of the system to first recognize and then label the proteins to be degraded with a poly-ubiquitin (Ub) chain. It is fairly assumed that the recognition of the substrate is accomplished by the numerous E3 ligases present in mammalian cells. However, most E3s do not possess any catalytic activity and E2 enzymes may be more than simple Ub-providers for E3s since they are probably important actors in the ubiquitination machinery. Surprisingly, most authors have tried to characterize E3 substrates, but the exact role of E2s in muscle protein degradation is largely unknown. A very limited number of the 35 E2s described in humans have been studied in muscle protein breakdown experiments and the vast majority of studies were only descriptive. We review here the role of E2 enzymes in skeletal muscle and the difficulties linked to their study and provide future directions for the identification of muscle E2s responsible for the ubiquitination of contractile proteins.
    Frontiers in Physiology 03/2015; 6:59. DOI:10.3389/fphys.2015.00059 · 3.53 Impact Factor
  • Source
    • "Gene ontology analysis revealed that the majority of these interactors had roles in 'establishment of localization', 'localization' and 'transport' (Fig. 1B). Network analysis (Fig. 1C) confirmed the presence of the known VPS35-interacting partners VPS26A and VPS26B, VPS29, TBC1D5 (Harbour et al., 2010), SNX27 (Steinberg et al., 2013) and all components of the actin polymerizing WASH complex (Derivery et al., 2009; Gomez and Billadeau, 2009; Harbour et al., 2010; Harbour et al., 2012; Jia et al., 2012; Hao et al., 2013). The lack of enrichment of the SNX-BAR components of the SNX-BAR–retromer is entirely consistent with the apparent low affinity of this interaction [the interaction of VPS29 with SNX1 has a K d of .150 "
    [Show abstract] [Hide abstract]
    ABSTRACT: Retromer is a protein assembly that orchestrates sorting of transmembrane cargo proteins into endosome-to-Golgi and endosome-to-plasma membrane transport pathways. Here, we have employed quantitative proteomics to define the interactome of human VPS35, the core retromer component. This has identified a number of new interacting proteins, including ankyrin-repeat domain 50 (ANKRD50), seriologically-defined colon cancer antigen 3 (SDCCAG3) and VPS9-ankyrin-repeat protein (VARP). Depletion of these proteins resulted in trafficking defects of retromer-dependent cargo, but differential and cargo specific effects suggested a surprising degree of functional heterogeneity in retromer-mediated endosome-to-plasma membrane sorting. Extending this, suppression of the retromer-associated WASH complex did not uniformly affect retromer cargo, thereby confirming cargo specific functions for retromer interacting proteins. Further analysis of the retromer-VARP interaction identified a role for retromer in endosome-to-melanosome transport. Suppression of VPS35 led to miss trafficking of the melanogenic enzymes, tyrosinase and tryrosine-related protein 1 (Tyrp1), establishing that retromer acts in concert with VARP in this trafficking pathway. Overall these data reveal hidden complexities in retromer-mediated sorting and open up new directions in our molecular understanding of this essential sorting complex.
    Journal of Cell Science 10/2014; 127(22). DOI:10.1242/jcs.156299 · 5.43 Impact Factor
  • Source
    • "Among those identified, the filament actin (F-actin) plays the most critical role in mediating dendritic spine morphology and function [26] [27]. Although it is unclear whether F-actin can be ubiquitinated, F-actin polymerization can be regulated indirectly by ubiquitination through the Wiskott-Aldrich syndrome protein (WASP) family member WASH-dependent mechanism [28]. Therefore, it would be an important topic to understand whether F-actin level and polymerization at synapses can also be regulated by ubiquitination-related mechanisms as that would likely in turn affect synapse plasticity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The ubiquitin proteasome system (UPS) is one of the principle mechanisms for the regulation of protein homeostasis in mammalian cells. In dynamic cellular structures such as neuronal synapses, UPS and protein translation provide an efficient way for cells to respond promptly to local stimulation and regulate neuroplasticity. The majority of research related to long-term plasticity has been focused on the postsynapses and has shown that ubiquitination and subsequent degradation of specific proteins are involved in various activity-dependent plasticity events. This review summarizes recent achievements in understanding ubiquitination of postsynaptic proteins and its impact on synapse plasticity and discusses the direction for advancing future research in the field.
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 08/2014; 1843(12). DOI:10.1016/j.bbamcr.2014.08.006 · 5.02 Impact Factor
Show more