Bead aggregation assays were performed as previously described (Wang et al., 2003 (link), 2005 (link); Tang et al., 2016 (link)). In brief, purified GRASP65 full-length or N-terminal fragment was coupled onto the surface of Dynabeads M-500 Subcellular or M-450 Tosylactivated (Invitrogen). The coupled beads were then incubated with IC or purified proteins as indicated in the presence of an ARS at 37°C with rotation for 1 h. The beads were then submitted for imaging and quantitation. Random phase contrast digital images (10–20) of each sample were captured using Zeiss Observer Z1 epifluorescence microscope with a 10× lens. Images from three independent experiments were analyzed using MATLAB7.4 software to determine the surface area of objects, which was used to calculate the number of beads in the clusters. Aggregates were defined as those with six or more beads. For immunodepletion of DjA1, IC prepared from HeLa S3 cells (Rabouille et al., 1995 ) was incubated with nonspecific control IgG or DjA1 antibody (ProteinTech) and protein A-agarose beads at 4°C overnight. The beads were pelleted, and supernatant was analyzed by Western blotting of DjA1.