JIP3 interacts with dynein and kinesin-1 to regulate bidirectional organelle transport

Abstract

The MAP kinase and motor scaffold JIP3 prevents excess lysosome accumulation in axons of vertebrates and invertebrates. How JIP3’s interaction with dynein and kinesin-1 contributes to organelle clearance is unclear. We show that human dynein light intermediate chain (DLIC) binds the N-terminal RH1 domain of JIP3, its paralog JIP4, and the lysosomal adaptor RILP. A point mutation in RH1 abrogates DLIC binding without perturbing the interaction between JIP3’s RH1 domain and kinesin heavy chain. Characterization of this separation-of-function mutation in Caenorhabditis elegans shows that JIP3–bound dynein is required for organelle clearance in the anterior process of touch receptor neurons. Unlike JIP3 null mutants, JIP3 that cannot bind DLIC causes prominent accumulation of endo-lysosomal organelles at the neurite tip, which is rescued by a disease-associated point mutation in JIP3’s leucine zipper that abrogates kinesin light chain binding. These results highlight that RH1 domains are interaction hubs for cytoskeletal motors and suggest that JIP3–bound dynein and kinesin-1 participate in bidirectional organelle transport.

Molecular mechanism of dynein-dynactin activation by JIP3 and LIS1

“…Instead its three main sites of interaction all involve the dynein tails (Figure 3A). The JIP3 N-terminal RH1 domain shows extra density (Figure 3B) consistent with the presence of DLIC C-terminal helices (Celestino et al 2022). Notably both sides of the RH1 domain are occupied suggesting two DLIC C-termini are bound.…”

“…Initially we expressed and purified full length JIP3, however, were unable to observe any activation of dynein motility in vitro (data not shown). As other full-length dynein adaptors are known to be autoinhibited (Hoogenraad et al 2003, d’Amico et al 2022) we generated a JIP3 truncation (1-185) consisting of just the RH1 domain and LZI coiled coil (Celestino et al 2022). We performed pull down experiments from pig brain lysates and immunoblotted for the p150 Glued component of dynactin and the dynein intermediate chain (DIC) (Figure 1B).…”

“…More distant relatives include RILP, RILPL1 and RILPL2. RILP directly binds to the small GTPase Rab7 (Wu et al 2005), recruits dynein/dynactin to late endosomes and lysosomes (Jordens et al 2001) and interacts with the DLIC C-terminus (Schroeder et al 2014, Celestino et al 2022). Unlike JIP3, we have not yet been able to detect dynein activation with RILP, but this may be due to not yet finding a construct in which autoinhibition mechanisms are removed.…”

“…5 µL aliquots were flash frozen and stored at -80°C. JIP31-185 construct (Celestino et al 2022) was expressed and purified as previously described (Celestino et al 2022). Briefly, the protein fragment from mouse cDNA was inserted into a 2CT vector [N-terminal 6xHis::maltose binding protein (MBP) followed by a TEV protease cleavage site and Cterminal Strep-tag II].…”

“…JIP3, its paralog JIP4 and the related RILP proteins (RILP, RILPL1 and RILPL2) share a similar architecture (Vilela et al 2019). At their N-termini is an RH1 (RILP Homology 1) domain which, with the exception of RILPL2, binds the DLIC C-terminus (Celestino et al 2022). Like other dynein adaptors, the RH1 domain is followed by a stretch of coiled coil, but unlike other adaptors this region is too short to span the length of dynactin (Reck-Peterson et al 2018).…”