Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding

The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L-) domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1). It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV), where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.     

SCFFBXL¹⁵ regulates BMP signalling by directing the degradation of HECT-type ubiquitin ligase Smurf1

Smad ubiquitination regulatory factor 1 (Smurf1), an homologous to E6AP C-terminus (HECT)-type E3 ubiquitin ligase, performs a crucial role in the regulation of the bone morphogenetic protein (BMP) signalling pathway in both embryonic development and bone remodelling. How the stability and activity of Smurf1 are negatively regulated remains largely unclear. Here, we report that F-box and LRR domain-containing protein 15 (FBXL15), an F-box protein of the FBXL family, forms an Skp1-Cullin1-F-box protein-Roc1 (SCF)(FBXL15) ubiquitin ligase complex and targets Smurf1 for ubiquitination and proteasomal degradation. FBXL15, through its leucine-rich repeat domain, specifically recognizes the large subdomain within the N-lobe of the Smurf1 HECT domain and promotes the ubiquitination of Smurf1 on K355 and K357 within the WW-HECT linker region. In this way, FBXL15 positively regulates BMP signalling in mammalian cells. Knockdown of fbxl15 expression in zebrafish embryos by specific antisense morpholinos causes embryonic dorsalization phenocoping BMP-deficient mutants. Injection of FBXL15 siRNAs into rat bone tissues leads to a significant loss of bone mass and decrease in bone mineral density. Collectively, our results demonstrate that Smurf1 stability is suppressed by SCF(FBXL15)-mediated ubiquitination and that FBXL15 is a key regulator of BMP signalling during embryonic development and adult bone formation.     

The WW1 Domain Enhances Autoinhibition in Smurf Ubiquitin Ligases

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with ~70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways.     

The HECT domain ligase itch ubiquitinates endophilin and localizes to the trans-Golgi network and endosomal system

Endophilin A1 is an SH3 domain-containing protein functioning in membrane trafficking on the endocytic pathway. We have identified the E3 ubiquitin ligase itch/AIP4 as an endophilin A1-binding partner. Itch belongs to the Nedd4/Rsp5p family of proteins and contains an N-terminal C2 domain, four WW domains and a catalytic HECT domain. Unlike other Nedd4/Rsp5p family members, itch possesses a short proline-rich domain that mediates its binding to the SH3 domain of endophilin A1. Itch ubiquitinates endophilin A1 and the SH3/proline-rich domain interaction facilitates this activity. Interestingly, itch co-localizes with markers of the endosomal system in a C2 domain-dependent manner and upon EGF stimulation, endophilin A1 translocates to an EGF-positive endosomal compartment where it colocalizes with itch. Moreover, EGF treatment of cells stimulates endophilin A1 ubiquitination. We have thus identified endophilin A1 as a substrate for the endosome-localized ubiquitin ligase itch. This interaction may be involved in ubiquitin-mediated sorting mechanisms operating at the level of endosomes.