Clathrin Adaptor GGA1 Polymerizes Clathrin into Tubules

Abstract

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.

Application of Gaussia luciferase in bicistronic and non-conventional secretion reporter constructs

“…GGAs are a family of monomeric clathrin adaptors that mediate the sorting of transmembrane cargo from the trans-Golgi network to late endosomes [48]. GGA1 can directly interact with clathrin [49-51], AP1 [52,53], ARF [54], Rabaptin-5 [55,56] and with a variety of cargo molecules [57], and is required for sorting of the lysosomal targeting receptor, mannose-6-phosphate receptor [48,52]. It has also been observed that overexpression of GGA3 inhibits retrovirus assembly [58], and that the molecule interacts with TSG101, an ubiquitin-binding component of the multivesicular body that is a key regulator of HIV budding.…”

The structure of a COPII tubule

“…Thus, the Sec13/31 tubule is well suited for binding Sec23/24 and a tubular membrane despite blocking some of the Sec23/24 binding sites on individual Sec13/31 heterotetramers. Interestingly, in the endocytic pathway, clathrin has also been shown to polymerize into tubules in the presence of the GGA adaptor molecule (Zhang et al, 2007). Analogously, it is possible in the COPII system that Sec23/Sec24, in combination with TANGO1 and procollagen, promote the formation of a Sec13/31 tubular carrier in vivo, though this hypothesis would be quite difficult to test experimentally.…”

Lysosomal protein trafficking in Giardia lamblia common and distinct features

“…Instead, uncharacteristic coated pits were seen in close association with the PVs (10), suggesting that a distinct arrangement of clathrin might occur in this parasite. It is also possible that, as was observed in vitro (35), clathrin may be organized in a hexagonal array, forming tubes instead of vesicles. Similar to TCs in HeLa cells (26), the tubules might not break down into CCVs en route to PVs in Giardia.…”