Membrane Deformation Ability of the Ankyrin Repeat and KH Domain-Containing Protein 1 (ANKHD1) and Its Involvement in the Early Endosome Enlargement

Abstract

Ankyrin-repeat domains (ARDs) are conserved in large numbers of proteins. ARDs are composed of ankyrin repeats (ANKs). The number of repeats varies among ARDs, and the sequence of ANKs often adopt curved structures reminiscent of the Bin-Amphiphysin-Rvs (BAR) domain, which is the dimeric membrane scaffold for membrane tubulation. BAR domains sometimes have amphipathic helices that enable the membrane scission for vesicle formation. Whether ARD-containing proteins exhibit similar membrane deformation and scission had been unclear. We found that the ARD of ankyrin repeat and KH domain-containing protein 1 (ANKHD1) dimerize and efficiently deform membranes into tubules and vesicles. ANKHD1 contains 25 ANKs that are divided into two groups; the first 15 ANKs and the latter 10 ANKs. We found that the first 15 ANKs can form a dimer, and the latter 10 ANKs enable membrane tubulation and vesiculation. These 10 ANKs were shown to have an adjacent amphipathic helix and were predicted to have a curved structure with a positively charged surface, analogous to BAR domains. Mutations of the positively charged amino acid residues and the deletion of the amphipathic helix abolished the membrane vesiculation ability. Interestingly, the dimeric 25 ANKs displayed significantly higher vesiculation ability than the 10 ANKs. Knockdown and localization of ANKHD1 suggested its involvement in the negative regulation of the enlargement of early endosomes. These results indicate that ANKHD1 causes vesiculation of the early endosomal membrane in a manner that is similar to the BAR domain protein.