The direct inter- and intramolecular interactions of LAMTOR and BORCΔBLOC-1 detected by in vitro chemical cross-linking. (A) Cross-linking of in vitro copurified LAMTOR (LAMTOR1Δ1-20, LAMTOR2, LAMTOR3, LAMTOR4, and LAMTOR5) and BORCΔBLOC-1 (lyspersin, myrlysin, KxDL1, MEF2BNB, and diaskedin) complexes with 0.2 mM BS3 results in a higher molecular weight band (arrow) detected by anti-lyspersin and anti-LAMTOR2 antibodies. (B) List of unique cross-links identified by mass spectrometry. Recombinant protein complexes were purified and cross-linked as described in A. The cross-linked proteins were digested and the tryptic peptides analyzed by mass spectrometry. Annotated are the cross-linked proteins, relevant peptide sequences, the lysines being cross-linked and the total number of identified peptide spectra matched (PSM). (C) The N terminus of LAMTOR2 cross-links with K224 of lyspersin (closest lysine to PRR); myrlysin and KxDL1 cross-link with the C-domain of lyspersin. The thickness of the lanes in the graphical representation corresponds to the number of cross-links identified in B.