News and Publications

New Insights Into the Regulation of Clathrin-Mediated Endocytosis

Clathrin-mediated endocytosis is essential for the efficient uptake of nutrients and other macromolecules into cells and for the regulation of signaling by cell-surface receptors. The process occurs at clathrin-coated pits, which concentrate receptor-ligand complexes, deform the membrane, invaginate, and eventually pinch off, forming clathrin-coated vesicles (CCVs). Clathrin is a self-assembling molecule that forms a curved polygonal lattice to drive membrane invagination.

Adaptor protein-2 (AP2) complexes are composed of 4 subunits: α, ß, µ2, and <font face="symbol">s</font face>2. The complexes are targeted to the plasma membrane through protein and lipid interactions involving the α subunit. Interactions between the ß subunit and clathrin trigger clathrin assembly, forming coated pits. The µ2 subunit interacts directly with tyrosine-based sorting motifs on the cytoplasmic tails of surface receptors to concentrate the receptors into the assembling coated pit. AP2 complexes also interact with several accessory proteins, perhaps coordinating the activities of the proteins in receptor-mediated endocytosis.

Taking a biochemical approach, we developed and use cell-free assays that faithfully reconstitute discrete events in clathrin-mediated endocytosis to discover new components of the endocytic machinery and to probe the hierarchy of interactions leading to coat assembly, cargo selection, vesicle budding, membrane fission, and CCV uncoating. Our results this past year revealed some surprises about how CCV formation is regulated.

Last year we discovered a new kinase, called adaptor-associated kinase-1, that phosphorylates the AP2 complex and regulates interactions of the complex with cargo molecules in vitro. We confirmed a role for this kinase in regulating AP2 function in vivo by using an adenoviral expression system to overexpress wild-type and mutant forms of the kinase in culture cells. However, we discovered that despite the disruption of AP2 function in these cells, clathrin-coated pits assembled normally and another cargo receptor, the receptor for epidermal growth factor, was efficiently internalized. These data suggest that AP2 complexes may not play an essential central role in controlling the assembly of coated pits, as previously thought, but may be more involved in coordinating specific cargo selection with the formation of CCVs.

These results are consistent with findings from a new in vitro assay that we developed for cargo selection and CCV formation that involves the use of highly purified plasma membrane sheets from rat liver. In this assay, CCV formation and the selective uptake of cargo receptors require clathrin, dynamin, and other cytosolic factors, but AP2 complexes are not a limiting component, as would have been expected. We are confident that this new assay will allow us to identify the central machinery involved in CCV formation and to explore the fundamental mechanisms that govern the formation.

The GTPase dynamin is a major regulator of endocytic formation of CCVs. Dynamin has several biochemical properties that distinguish it from more conventional GTPases that act as regulatory molecules. It has a low affinity for GTP, it self-assembles into rings and helical stacks of rings, and it encodes its own GTPase-activating protein domain, the GTPase effector domain, that is activated upon self-assembly. Self-assembly stimulates the GTPase activity of dynamin, in a manner that depends on the GTPase effector domain, approximately 100-fold.

Two prevailing models for dynamin function in endocytosis exist, both of which, ironically, we proposed. The first suggests that dynamin self-assembles into collarlike structures at the necks of deeply invaginated coated pits. Upon self-assembly, rapid GTP hydrolysis causes a concerted conformational change in the assembled dynamin that constricts the structure and drives membrane fission and vesicle detachment. In this model, dynamin functions as a mechanochemical enzyme, and assembly-stimulated GTP hydrolysis drives a power stroke-like conformational change that actively drives membrane fission.

The second model posits that dynamin functions the same as all other members of the GTPase superfamily, as a regulatory GTPase that recruits and/or activates downstream effectors required for the maturation of coated pits and the formation of vesicles. Dynamin self-assembly at the necks of coated pits serves as a geometric sensor that signals termination of the reaction and enables recycling of the components of the endocytic machinery. In this model, dynamin functions as a kinetic timer, monitoring and/or directing molecular events at the coated pit, and assembly-stimulated GTP hydrolysis resets the clock.

New results support a new model that incorporates both aspects of these two extremes. We found that dynamin self-assembly and assembly-stimulated GTP hydrolysis are required for clathrin-mediated endocytosis but that assembly-stimulated GTP hydrolysis is not rate limiting for this process. Further studies, aided by our new dynamin-dependent cell-free assay for vesicle formation, should help define the unique role of dynamin in the formation of endocytic vesicles.

Publications

Chang, H.C., Newmyer, S.L., Hull, M.J., Ebersold, M., Schmid, S.L., Mellman, I. Hsc70 is required for endocytosis and clathrin function in Drosophila. J. Cell Biol. 159:477, 2002.

Conner, S.D. Schmid, S.L. Regulated portals of entry into the cell. Nature 422:37, 2003.

Miwako, I., Schröter. T., Schmid, S.L. Clathrin- and dynamin-dependent coated vesicle formation from isolated plasma membranes. Traffic 4:376, 2003.

Schmid, S.L. Conventional and unconventional aspects of dynamin GTPases. In: G Proteins. Bradshaw, R., Dennis, E. (Eds.). Academic Press, San Diego, 2003, p. 763. Handbook of Cellular Signaling, Vol. 2.

Song, B.D., Schmid, S.L. A molecular motor or a regulator? Dynamin's in a class of its own. Biochemistry 42:1369, 2003.