Gamma-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation.

Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular gamma2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered gamma2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same gamma2-adaptor and that disruption of the HBV/gamma2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of gamma2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both gamma2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with gamma2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.

The vesicular acetylcholine transporter interacts with clathrin-associated adaptor complexes AP-1 and AP-2.

In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.

Structural modeling of ataxin-3 reveals distant homology to adaptins.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3.