Updated FTP location. In this study, pull down assays combined with mass spectrometry analysis were performed in Haloferax volcanii cells containing different LonB content to identify the repertoire of LonB interacting proteins. H. volcanii H26 and HVLON3 (Lon deficient mutant) were grown in Hv-Min medium and samples were taken at exponential (EXP) and stationary (ST) phase. The cells were treated with crosslinkers DSP (1 mM) for 2 h or formaldehyde (1%) for 10 min at room temperature. Crosslinking reactions were stopped and the cells were harvested, washed, and suspended in saline phosphate buffer containing protease inhibitors. The cells were disrupted, cell debris was removed, and membranes were pelleted and washed in the same buffer. Membranes were incubated in 1× PBS with 1% dodecyl maltoside (DDM) for 1 h at 42 °C and then subjected to immunoprecipitation with anti-NmLon polyclonal antiserum conjugated to Protein A−Sepharose beads CL-4B. As a negative control, in parallel, samples were immunoprecipitated with the non-specific anti-Nep antiserum corresponding to the extracellular N. magadii protease. After incubation with the antibodies, the bead slurry was added to the extracts, gently agitated overnight at 4 °C and washed three times with 1× PBS containing ammonium hydrogen carbonate (AMBIC). On-bead digestion was performed by adding 10 μL of trypsin (10 ng/μL) in 100 mM AMBIC and incubated overnight at 37 °C with agitation. The peptide mix was recovered and 5% (v/v) formic acid was added. The peptides were dried in Speedvac, suspended in 100 μL of Buffer A (0.1% formic acid in water), desalted by zip-tipping (C18 membranes) and analyzed by MS, as described in Cerletti et al. (2018). Proteins were identified and quantified using MaxQuant version 1.5.3.17 with LFQ algorithm. The parameters were set as described in Cerletti et al. (2018); threshold Andromeda scores for unmodified and modified peptides were 0 and 40, respectively.