BORC–Ragulator interactions are mediated by the lyspersin DUF2365 domain and LAMTOR2. (A) Predicted coiled coils (Coils server, embnet.vital-it.ch/software/COILS_form.html) and consensus secondary structure (NPS@, https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html) of human lyspersin. Blue, α-helix; red, β-sheet; magenta, random coil structure. Amino acid numbers are indicated. (B) Comparison of the domain organization of lyspersin in different species. DUF2365 comprises two predicted folded segments designated CE1 and CE2. Key residues in CE1 and CE2 and amino acid numbers in the human protein are indicated. H. sapiens, Homo sapiens; X. tropicalis, Xenopus tropicalis; D. rerio, Danio rerio; C. elegans, Caenorhabditis elegans; D. melanogaster, Drosophila melanogaster. (C) GFP or GFP-tagged full-length, truncated, or mutated lyspersin (Lysp) were expressed by transfection into lyspersin-KO HeLa cells. Cells were extracted in detergent and subjected to immunoprecipitation with GFP-Trap beads. Endogenous myrlysin, LAMTOR1, LAMTOR4, and transgenic GFP were detected by immunoblotting (IB). The positions of molecular mass markers (in kilodaltons) are indicated. Arrows indicate the undegraded GFP-fusion proteins. (D and E) Y2H analysis of the interaction of lyspersin constructs fused to Gal4-BD (left) and BORC or Ragulator subunits fused to Gal4-AD (top). “, lyspersin. (F) Structural models generated with UCSF chimera (Pettersen et al., 2004) showing a potential lyspersin-binding site on the reported structure of LAMTOR2-LAMTOR3 (PDB code: 1VET [Kurzbauer et al., 2004]). (Left) Ribbon diagram showing amino acid residues at the potential binding site. (Right) Hydrophobicity surface representation highlights the hydrophobicity of the potential binding site (red patch at the top). Blue for the hydrophilic, to white, and to red for the hydrophobic residues. (G) Y2H analysis of the interaction of lyspersin or LAMTOR3 fused to Gal4-BD (left) and LAMTOR2 or LAMTOR2 mutants fused to Gal4-AD (top).