Members of the RME-1/mRme-1/EHD1 protein family have recently been shown to function in the recycling of membrane proteins from recycling endosomes to the plasma membrane. RME-1 family proteins are normally found in close association with recycling endosomes and the vesicles and tubules emanating from these endosomes, consistent with the proposal that these proteins directly participate in endosomal transport. RME-1 family proteins contain a C-terminal EH (eps15 homology) domain thought to be involved in linking RME-1 to other endocytic proteins, a coiled-coil domain thought to be involved in homo-oligomerization and an N-terminal Ploop domain thought to mediate nucleotide binding. In the present study, we show that both Caenorhabditis elegans and mouse RME-1 proteins bind and hydrolyze ATP. No significant GTP binding or hydrolysis was detected. Mutation or deletion of the ATP-binding P-loop prevented RME-1 oligomerization and at the same time dissociated RME-1 from endosomes. In addition, ATP depletion caused RME-1 to lose its endosome association in the cell, resulting in cytosolic localization. Taken together, these results indicate that ATP binding is required for oligomerization of mRme-1/EHD1, which in turn is required for its association with endosomes.

Clathrin and clathrin adaptors on clathrin-coated pits exchange with cytosolic clathrin and clathrin adaptors in vivo. This exchange might require the molecular chaperone Hsc70 and J-domain-protein auxilin, which, with ATP, uncoat clathrin-coated vesicles both in vivo and in vitro. We find that, although Hsc70 and ATP alone could not uncoat clathrin-coated pits, further addition of auxilin caused rapid uncoating of clathrin but not AP2 and epsin. By contrast, cytosol uncoats clathrin, AP2 and epsin from pits in permeabilized cells, and, concomitantly, these proteins in the cytosol rebind to the same pits, establishing that, like in vivo, these proteins exchange in permeabilized cells. Dissociation and exchange of clathrin in permeabilized cells can be prevented by inhibiting Hsc70 activity. The presence of clathrin-exchange in the permeabilized system substantiates our in vivo observations, and is consistent with the view that Hsc70 and auxilin are involved in the clathrin-exchange that occurs as clathrin-coated pits invaginate in vivo.

GGAs, a class of monomeric clathrin adaptors, are involved in the sorting of cargo at the trans-Golgi network of eukaryotic cells. They are modular structures consisting of the VHS, the GAT, hinge, and GAE domains, which have been shown to interact directly with cargo, ARF, clathrin, and accessory proteins, respectively. Previous studies have shown that GGAs interact with clathrin both in solution and in the cell, but it has yet been shown whether they assemble clathrin. We find that GGA1 promoted assembly of clathrin with complete assembly achieved when one GGA1 molecule is bound per heavy chain. In the presence of excess GGA1, we obtained the unusual stoichiometry of five GGA1s per heavy chain, and even at this stoichiometry the binding was not saturated. The assembled structures were mostly baskets, but ϳ10% of the structures were tubular with an average length of 180 ؎ 40 nm and width of ϳ50 nm. The truncated GGA1 fragment consisting of the hinge؉GAE domains bound to clathrin with similar affinity as the full-length molecule and polymerized clathrin into baskets. Unlike the fulllength molecule, this fragment saturated the lattices at one molecule per heavy chain and assembled clathrin only into baskets. The separated hinge and GAE domains bound much weaker to clathrin than the intact molecule, and these domains do not significantly polymerize clathrin into baskets. We conclude that clathrin adaptor GGA1 is a clathrin assembly protein, but it is unique in its ability to polymerize clathrin into tubules.