Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization

Abstract

HIV-1 Nef is an important pathogenic factor for HIV/AIDS pathogenesis. Studies have shown that the association of Nef with the inner leaflet of the plasma membrane and with endocytic and perinuclear vesicles is essential for most activities of Nef. Using purified recombinant proteins in pull-down assays and by co-immunoprecipitation assays we demonstrate that Nef binds directly and specifically to all GABARAP family members, but not to LC3 family members. Based on nuclear magnetic resonance (NMR) experiments we showed that Nef binds to GABARAP via two surface exposed hydrophobic pockets. S53 and F62 of GABARAP were identified as key residues for the interaction with Nef. During live-cell fluorescence microscopy an accumulation of Nef and all GABARAP family members in vesicular structures throughout the cytoplasm and at the plasma membrane was observed. This plasma membrane accumulation was significantly reduced after knocking down GABARAP, GABARAPL1 and GABARAPL2 with respective siRNAs. We identified GABARAPs as the first known direct interaction partners of Nef that are essential for its plasma membrane localization.

Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations

“…On the other hand, we observed more inter-microcluster H-bonds in HP2 of GABARAP proteins (21) than LC3 (19), indicating tighter packing in GABARAP proteins. Previous reports also suggested similar observations where two-dimensional (2D) 1H-15N-heteronuclear single quantum coherence (HSQC) spectra showed HP2 to be more affected than HP1 on HIV-1 Nef binding to GABARAP [45]. It has also been reported that KBTBD6 engages with the bulkier residues of HP2 in GABARAP proteins, thus, forming tight-complex contributing to high binding affinity [25].…”

“…In a high-throughput study, LC3/GABARAP subfamilies display interactome differences where around one-third of binding partners were found to be specific for LC3 subfamily, one-third for GABARAP and one-third were found to be common for both groups [18]. At the molecular level, many reports have identified unique binders to HsAtg8 orthologs [21,[23][24][25][26][27][28][29][30]45]. We, therefore, surmised that molecular differences between bound complexes of human Atg8 orthologs would entail underlying mechanisms into their selectivity.…”

“…The importance of these residues has been established by mutagenesis experiments whereby alanine mutant of F52 and Y49 abrogates LC3 binding to SQSTM1/p62 [36], GABARAP binding to NBR1 [26] and BNIP3L/Nix (BCL2 interacting protein 3 like) [21], respectively. In addition, other residues around HP1 and HP2 like Y25, V29, K46 and L55 in GABARAP proteins have been reported to display specific binding to WDFY3/ALFY, KBTBD6 and HIV-1 Nef [24,25,45]. In addition, HP1, being more conserved than HP2 in Atg8 protein family [7], displayed no major differences in H-bonding pattern.…”

The Atg8 Family of Proteins—Modulating Shape and Functionality of Autophagic Membranes

“…Beyond their well-established role in autophagy, Atg8 proteins may be implicated in aging processes in additional, previously unanticipated ways. Results from our laboratory suggest that mammalian GABARAP subfamily members are required for secretion processes generating extracellular vesicles ( Boeske et al, 2017 ). If produced by challenged cells, such vesicles may carry altered macromolecules serving as damage-associated molecular patterns (DAMPs), or signaling molecules such as microRNAs, possibly contributing to a chronic low-grade auto-inflammatory state characteristic of organismal aging ( Franceschi et al, 2017 ) as well as cell-to-cell propagation of a senescent phenotype ( Eitan et al, 2016 ).…”

Human ubiquitin-like proteins as central coordinators in autophagy

“…Thus, most GABARAP interaction partners have been found to bind GABARAPL1 and -L2 as well. For example, all three GABARAPs interact with ALFY in aggrephagy, with TRIM5a in antiviral xenophagy and with HIV-1 Nef in viral budding [45,54,79]. Whether one of the three proteins is the major interaction partner remains, however, unclear.…”