Membrane recruitment of endocytic proteins is regulated by cooperativity

Clathrin-mediated endocytosis is a highly orchestrated process which involves over 40 proteins, many of which are transiently recruited to the plasma membrane. A bottom up approach to reconstitute cooperative membrane binding and membrane scission of proteins involved in the process was used to study this poorly understood problem.

HFSP Long-Term Fellow Michael Meinecke and colleagues
authored on Wed, 27 March 2013

Clathrin mediated endocytosis (CME) is the predominant cellular pathway for the uptake of extracellular nutrients, receptor internalization, the retrieval of synaptic vesicle proteins and the uptake of lipids to regulate the content and area of the plasma membrane. Decades of intense research led to the current model of CME that starts with the interaction of cell surface receptors with adaptor proteins at random sites on the plasma membrane to stabilize nascent clathrin-coated pits. Maturing pits acquire cargo and invaginate via the coordinated action of curvature inducing proteins and clathrin polymerization. The neck of the deeply invaginated clathrin-coated pit is severed and a mature clathrin-coated vesicle is released. The whole process is highly regulated and involves the concerted action of some 40 proteins. Many of these proteins are transiently recruited to the plasma membrane with a high spatial and temporal fidelity.

The cover page from the Journal of Biological Chemistry (2013) 288. The background shows giant unilamellar vesicles labeled with a red fluorescent dye as a model system to reconstitute endocytic events. The foreground shows an abstract cartoon illustrating the cooperative membrane binding of dynamin and BAR (BIN/amphiphysin/Rvs) domain-containing proteins.

Two major players in CME are BAR domain-containing proteins and the large GTPase dynamin. BAR (BIN/Amphiphysin/Rvs) domain-containing proteins, like endophilin and amphiphysin, consist of an N-terminal BAR domain and a C-terminal SH3 domain. Via their BAR domains, these proteins have the ability to bind to membranes and to induce membrane curvature. The SH3 domain is a protein-protein interaction domain with which endophilin and amphiphysin can, for example, interact with dynamin. The large GTPase dynamin is a mechanochemical enzyme that catalyzes membrane scission at the neck regions of mature clathrin-coated pits. Although it is known that dynamin as well BAR domain-containing proteins can bind to membranes and interact with each other, it is poorly understood how the plasma membrane recruitment of these proteins can be regulated in vivo. Also, the effect that the interaction of dynamin with endophilin and amphiphysin has on dynamin-dependent membrane scission is only vaguely understood.

HFSP Long-Term Fellow, Michael Meinecke, now proposes an explanation for these questions. Using model membranes to reconstitute membrane binding with real time resolution, it was found that the recruitment of dynamin to membranes is slow in the absence of other proteins. The presence of BAR domain-containing endophilin or amphiphysin significantly accelerates this process. He discovered that, surprisingly, membrane binding of endophilin and amphiphysin relies on the interaction with dynamin. It was found that an auto-inhibition mechanism of these proteins in which the SH3 domain most likely binds back to the BAR domain prevents membrane binding. This auto-inhibition is released upon binding of the SH3 domain to the proline rich domain of dynamin, leading to cooperative membrane binding of dynamin and endophilin and amphiphysin. In addition, GTP dependent membrane scission by dynamin is highly increased in the presence of endophilin or amphiphysin. Consistent with the in vitro data, dynamin recruitment to the plasma membrane in cells was strongly reduced in the absence of endophilin and amphiphysin. Furthermore, amphiphysin depletion severely inhibited clathrin-mediated endocytosis.

Reference

Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission. Michael Meinecke*, Emmanuel Boucrot, Gamze Camdere, Wai-Chin Hon, Rohit Mittal, Harvey T. McMahon*. J. Biol. Chem. (2013) 288 , 6651-6661 (*corresponding authors)


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