Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the Cterminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus−cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.

Ubiquitin-conjugating enzymes (E2s) have a dominant role in determining which of the seven lysine residues of ubiquitin is used for polyubiquitination. Here we show that tethering of a substrate to an E2 enzyme in the absence of an E3 ubiquitin ligase is sufficient to promote its ubiquitination, whereas the type of the ubiquitin conjugates and the identity of the target lysine on the substrate are promiscuous. In contrast, when an E3 enzyme is introduced, a clear decision between mono-and polyubiquitination is made, and the conjugation type as well as the identity of the target lysine residue on the substrate becomes highly specific. These features of the E3 can be further regulated by auxiliary factors as exemplified by MDMX (Murine Double Minute X). In fact, we show that this interactor reconfigures MDM2-dependent ubiquitination of p53. Based on several model systems, we propose that although interaction with an E2 is sufficient to promote substrate ubiquitination the E3 molds the reaction into a specific, physiologically relevant protein modification.Targeting of most substrates to the 26 S proteasome requires covalent marking with polyubiquitin chains. Protein ubiquitination is a multistep process accomplished by the concerted action of three enzymes. The reaction begins with the ubiquitin-activating enzyme (E1), which initially adenylates the C-terminal glycine of ubiquitin and then forms a thioester bond between the activated glycine residue and a cysteine residue in its active site. Subsequently, a ubiquitin-conjugating enzyme (E2) acquires the activated ubiquitin through a trans-thioesterification reaction. Finally, a ubiquitin-protein ligase (E3) recruits a target protein and guides the transfer of the activated ubiquitin from the E2 to the substrate (1-3). Ubiquitin transfer from the E2 enzyme to the substrate is catalyzed directly by really interesting new gene (RING) 3 finger-containing E3s or indirectly when a homologous to E6-AP C terminus (HECT) domain E3 is mediating the transfer (4). Several forms of ubiquitination have been identified (5). Monoubiquitination or multiple monoubiquitinations are referred to as the conjugation of single or multiple ubiquitin moieties to distinct lysine residues on the substrate. These forms of ubiquitination were implicated in various cellular pathways, which include endocytosis and sorting of proteins to different cellular compartments (6, 7), as well as in several cases of proteasomal activity, such as the processing of the p105 precursor of the transcription regulator NF-B (8). However, polyubiquitination is the most common post-translational modification of proteins destined for degradation (9).In polyubiquitination assembly, ubiquitin conjugation was originally thought to be repeated in a cyclic manner whereby in each step a new moiety of ubiquitin is linked to one of the lysine residues of the previously conjugated ubiquitin. However, in view of recent findings, several alternative mechanisms have been proposed (10). Li et al. (11) demonstrated in a recon...