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Dynamin and Other Factors Regulating Receptor-Mediated Endocytosis

S.L. Schmid, H. Damke, L.M. Fujimoto, L.A. Hannan, M. Lötscher, A.B. Muhlberg , S.L. Newmyer, S. Sever, F. Simpson, L.J. Terlecky

Receptor-mediated endocytosis is essential for efficient uptake of nutrients, growth hormones, and immune complexes into cells. Many viruses also use this pathway to invade cells or to remodel the plasma membrane and avoid detection. The process occurs at specialized regions on the plasma membrane, called coated pits.

The coat in coated pits is assembled from 2 protein complexes (Fig. 1). Molecules of adaptor protein 2 (AP2) bind to unidentified docking proteins on the plasma membrane. This binding initiates coat formation (step 1) by triggering the subsequent assembly of clathrin into a curved protein lattice (step 2) that pulls the membrane inward, forming a deep pocket. AP2s also interact directly with the cytoplasmic tails of cell-surface receptors to affect the concentration of receptors in the deepening coated pit. A third protein, the GTPase dynamin, is targeted to the AP2 and clathrin coat (step 3). Upon binding GTP, dynamin self-assembles into a small spiral structure that forms a "collar" at the neck of an invaginated coated pit (step 4).

We have speculated that the coordinated hydrolysis of GTP by the assembled dynamin polypeptides causes a concerted conformational change that tightens the collar and facilitates detachment of a sealed coated vesicle (step 5). Before the coated vesicle can deliver its concentrated cargo to the appropriate intracellular destination, the coat proteins must be shed. Shedding is a 2-step reaction. Clathrin is first released (step 6) through the action of hsc70, the so-called uncoating ATPase. A subsequent reaction (step 7) drives release of AP2.

During the past several years, we have developed cell-free assays that faithfully reconstitute each of these 7 steps. We are using these assays to detect new proteins required for receptor-mediated endocytosis and to elucidate the mechanism of action of known proteins involved in this complex set of reactions.

FACTORS REQUIRED FOR COAT ASSEMBLY AND VESICLE BUDDING

Separate, but related, assays with perforated cells efficiently reconstitute early and late events in the formation of coated vesicles. As expected, early events require adaptor proteins, whereas late events require dynamin. However, both assays also require additional components from the cytosol. We are fractionating cytosol to determine the components required for AP2-dependent coat assembly and for dynamin-dependent vesicle budding. The former activity appears to be GTPS sensitive, whereas the latter activity appears to require ATP hydrolysis. The 2 activities are present in different fractions of the cytosol, a finding that suggests the activities are distinct.

FACTORS REQUIRED FOR AP2 RELEASE

We have developed a new rapid and quantitative assay for AP2 release from purified coated vesicles and have established that this reaction requires both hsc70 and an as-yet unidentified cytosolic factor. The second factor appears to be an ATPase, and work is under way to identify this component. In addition, we are using mutants of hsc70 defective in discrete stages of the ATPase cycle to investigate the mechanism of hsc70-mediated clathrin release.

MECHANISM OF ACTION OF DYNAMIN

Our model for dynamin function requires that GTP hydrolysis by assembled dynamin molecules be highly coordinated. Moreover, we suggest that a concerted conformational change driven by this coordinated GTP hydrolysis would generate the force necessary (although not sufficient) for detachment of coated vesicles. How might this coordination be accomplished?

During the past 2 years, we established that dynamin GTPase activity is highly cooperative and that dynamin self-assembly triggers rapid rates of GTP hydrolysis. Last year, using limited proteolysis to dissect the 100-kD dynamin molecule, we detected a domain of about 13 kD, the GTPase effector domain (GED), located near the C-terminus that is required for efficient GTP hydrolysis by the N-terminal GTPase domain.

More recently we expressed both the N-terminal GTPase domain and GED, independently, in Escherichia coli and showed that the basal rate of GTP hydrolysis of the GTPase domain is stimulated approximately 8-fold by addition of GED. Strikingly, GED stimulates GTPase activity of intact dynamin almost 100-fold. Our data establish that GED both regulates dynamin self-assembly and functions as an intramolecular GTPase-activating protein that couples dynamin assembly to stimulated GTP hydrolysis. Mutagenesis studies have identified residues involved in these 2 activities. The small size of GED makes it amenable to structural analysis by nuclear magnetic resonance, and we hope to use this technique to identify the residues that directly interact with the GTPase domain and those that regulate higher order assembly reactions.

PUBLICATIONS

Benmerah, A., Lamaze, C., Bègue, B., Schmid, S.L., Dautry-Varsat, A., Cerf-Bensussan, N. AP-2/Eps15 interaction is required for receptor-mediated endocytosis. J. Cell Biol. 140:1055, 1998.

Hannan, L.A., Newmyer, S.L., Schmid, S.L. ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for hsc70. Mol. Biol. Cell 9:2217, 1998.

Llorente, A., Rapak, A., Schmid, S.L., van Deurs, B., Sandvig, K. Expression of mutant dynamin inhibits toxicity and transport of endocytosed ricin to the Golgi apparatus. J. Cell Biol. 140:553, 1998.

Schmid, S.L., Cullis, P.R. Endosome marker is fat not fiction. Nature 392:135, 1998.

Schmid, S.L., McNiven, M.A., DeCamilli, P. Dynamin and its partners: A progress report. Curr. Opin. Cell Biol. 10:504, 1998.