News and Publications

Molecular Mechanisms Governing Receptor-Mediated Endocytosis

S.L. Schmid, B.P. Ceresa, S.D. Conner, H. Damke, K.N. Fish, L.M. Fujimoto, A. Jones, M. Jost, S.L. Newmyer, B. Panda, S. Sever, S. Sholly, F. Simpson

Receptor-mediated endocytosis is essential for the efficient uptake of nutrients, growth hormones, and immune complexes into cells. The process occurs at specialized regions on the plasma membrane called coated pits. The coat is assembled from 2 protein complexes, adaptor proteins and clathrin. Adaptor proteins are targeted to the plasma membrane and trigger the subsequent assembly of clathrin into a curved polygonal lattice that pulls the membrane inward, forming a deep pocket. Adaptor proteins also interact directly with receptors to affect the concentration of receptors in coated pits. Coated pits continue to gain curvature and become deeply invaginated before membrane fission at their necks releases a coated vesicle carrying cargo into the cell.

Biochemical, "stage-specific" assays developed in our laboratory enable us to measure distinct events leading to the formation of coated vesicles both in intact cells and in perforated cell membranes. In perforated cells, we focus on early events leading to coat assembly. We identified domains of the adaptor protein 2 complex that are essential for assembling a functionally active coated pit. We also identified a cytosolic activity necessary for adaptor protein 2--mediated endocytosis and are purifying the factor responsible for this activity.

Receptor-mediated endocytosis is tightly regulated, and much of our recent work has focused on the large GTPase dynamin, a key regulator of receptor-mediated endocytosis. We developed dominant-negative mutants of dynamin that inhibit receptor-mediated endocytosis when overexpressed, and last year we described the first "activating" mutants of dynamin, which stimulate receptor-mediated endocytosis. Using our stage-specific biochemical assays in combination with morphologic analysis, we established that these novel activating mutants of dynamin accelerate the formation of constricted coated pits, a late and normally transient intermediate in vesicle formation. Our results suggest that dynamin plays a role in regulating the membrane morphogenesis required to form deeply invaginated and constricted coated pits. New classes of more potently activating mutants of dynamin are being designed to explore further the consequences of expression of mutant GTPases.

The mammalian genome encodes 3 isoforms of dynamin, which are expressed in a tissue-specific manner. In contrast, the genomes of Caenorhabditis elegans and Drosophila encode only a single dynamin isoform. Our results suggest that dynamin-1, the neuron-specific mammalian isoform of dynamin, is highly specialized to regulate the extremely rapid endocytic events at the synapse needed to recycle synaptic vesicles. We discovered that the ubiquitously expressed mammalian isoform, dynamin-2, has a distinct function: when slightly overexpressed in dividing cells, it induces p53-dependent apoptosis. Dynamin-2--dependent apoptosis requires the ability of the dynamin to bind GTP, suggesting that binding of dynamin-2 to GTP triggers a signaling cascade that activates p53. These results are especially intriguing because the only other dynamin-related proteins expressed in C elegans or Drosophila are 2 proteins that regulate membrane dynamics at the mitochondria. Taken together, our results suggest that members of the dynamin family may regulate membrane dynamics at critical locations in the cell (on the mitochondrial membrane and the plasma membrane) so that the dynamins can monitor the fidelity of these events, the membrane integrity, and cellular homeostasis. We are pursuing this hypothesis.

Once a vesicle is formed, the coat proteins are recycled, and the naked vesicle is released for fusion with a cellular "sorting depot" called the sorting endosome. The chaperonin hsc70 uncoats isolated coated vesicles in vitro. We developed dominant-negative mutants of hsc70 and established a role for this chaperonin in the uncoating reaction and endocytic trafficking in vivo.

PUBLICATIONS
Bark, S.J., Schmid, S.L., Hahn, K.M. A highly efficient method for site-specific modification of unprotected peptides after chemical synthesis. J. Am. Chem. Soc. 122:3567, 2000.

Ceresa, B.P., Schmid, S.L. Regulation of signal transduction by endocytosis. Curr. Opin. Cell Biol. 12:204, 2000.

Damke, H., Muhlberg, A.B., Sever, S., Sholly, S., Warnock, D.E., Schmid, S.L. Expression, purification and functional assays for self-association of dynamin-1. Methods Enzymol., in press.

Fish, K.N., Schmid, S.L., Damke, H. Evidence that dynamin-2 functions as a signal transducing GTPase. J. Cell Biol. 150:145, 2000.

Fujimoto, L.M., Roth, R., Heuser, J.E., Schmid, S.L. Actin assembly plays a variable but not obligatory role in receptor-mediated endocytosis. Traffic 1:161, 2000.

Muhlberg, A.B., Schmid, S.L. Domain structure and function of dynamin probed by limited proteolysis. Methods 20:475, 2000.

Schmid, S.L. Dynamin. In: Guidebook to Cytoskeletal and Motor Proteins. Vale, R., Kries, T. (Eds.). Oxford University Press, New York, 1999, p. 524.

Sever, S., Damke, H., Schmid, S.L. Dynamin:GTP controls the formation of constricted coated pits, the rate limiting step in clathrin-mediated endocytosis. J. Cell Biol. 150:1137, 2000.

Sever, S., Damke, H., Schmid, S.L. Garrotes, springs, ratchets and whips: Putting dynamin models to the test. Traffic 1:385, 2000.