Structural Basis of WHAMM to Regulate Golgi Membrane Transportation via Interacting with Both Microtubules and Membranes

WHAMM protein is a newly identified mammalian nucleation-promoting protein, and it is comprised of an N-terminal domain that mediates Golgi membrane association, a coiled-coil domain (CC) that binds to microtubule, and a WCA domain that stimulates Arp2/3-mediated actin polymerization. WHAMM protein plays key roles in both maintaining Golgi structure and facilitating anterograde membrane transport. The molecular mechanism for WHAMM to fulfill its functions remains unknown. We have used cryo-electron microscopy (cryo-EM) to investigate the structural basis of WHAMM's interaction with microtubules and membranes.Our in vitro pelleting assay shows that both full-length WHAMM protein and the CC domain have high binding affinity to Taxol-stabilized microtubules at around 360 nM. This is verified by Cryo-EM observation that both full-length WHAMM protein and CC assemble around microtubules within a few minutes of incubation. Image analysis of the micrographs indicates that the proteins assemble with stoichiometric binding to the alpha-beta tubulin heterodimers in the microtubule lattice. We performed three-dimensional reconstruction of the WHAMM and CC assemblies around microtubules and revealed that WHAMM binds to the outer surface of microtubules via direct interaction between its CC domain with tubulin dimer. The assembly of WHAMM onto microtubule exposes the N-terminal domain ready to bind membranes.We have further used fluorescence microscopy to demonstrate that vesicles made of lipids from liver extract can be recruited to microtubules in the presence of WHAMM in an in vitro assay. Cryo-EM of such a specimen verified the cross-bridge between the lipid vesicles and microtubules by WHAMM and showed that the interaction causes the vesicles to change their shapes along the microtubules.