Clathrin-mediated endocytosis

Clathrin-coated pits (CCPs) and vesicles were initially visualized as bristle coated vesicles and membranes during the electron microscopical analysis of mosquito oocytes taking up extracellular yolk proteins (Roth and Porter, 1964). During the forthcoming four decades, major structural and functional components of these

‘bristle’ coated membrane structures have been identified one by one (Pearse, 1975;

Keen et al., 1979), and the steps involved in early nucleation of coated pits to the final dissociation of the clathrin have been well characterized (Takei and Haucke, 2001 ).

CCPs assemble on cytosolic leaflets of the plasma membrane as well as the trans-Golgi network (TGN) membranes, and function in sorting of proteins into transport vesicles that eventually fuse with endosomes and lysosomes. A good analogy for sorting of cargo proteins to coated pits, generation of coated pits and fission of clathrin-coated vesicles would be an arm picking up some objects from the ground.

According to this analogy clathrin can be compared to the bones and skeleton of the arm, as its major function is that of a structural scaffold for the uptake event. Adapter proteins including AP2 (or AP1 in case of budding from the TGN) that recognize and deliver the membrane cargo proteins to CCPs can be compared to the hand in the same analogy. Just as a hand needs fingers for fine adjustment and recognition of

different objects, AP2 utilizes a plethora of accessory adapter proteins that recognize a variety of different cargo molecules. Finally, dynamin could be compared to the muscles, as its GTPase activity provides the energy for the final budding event.

There are also other factors that are essential for CME (i.e. uncoating factors) which do not fit into this analogy.

1.3.1. Sequence of events in clathrin-mediated endocytosis

CME can be roughly divided into 4 steps; i) nucleation, ii) bud formation, iii) fission, and iv) uncoating (Figure 1.3).

Clathrin-coated pit nucleation is mediated by adapter proteins and other endocytic accessory proteins (i.e. epsin, AP180) which form a dynamic network of protein-protein interactions. Endocytic accessory protein-proteins recognize membrane cargo proteins using the sorting motifs present in their cytosolic tails (Bonifacino and Traub, 2003) or attached mono-ubiquitin residues (Aguilar and Wendland, 2005; Chen and De Camilli, 2005; Sigismund et al., 2005). Binding of adapter proteins to cargo molecules initiates coated pit nucleation. For instance, during synaptic vesicle endocytosis, binding of AP2 to peptides bearing tyrosine-based endocytic sorting motifs stimulates their interaction with synaptotagmin 1 (Haucke and De Camilli, 1999; Haucke et al., 2000).

Membrane lipids also serve an active role in the coat nucleation event. Endocytic accessory proteins like AP2, AP180 or CALM and epsin are recruited to the plasma membrane during endocytosis by binding to certain plasma membrane lipids, in particular to PIP2 (Phosphatidylinositol (4,5)-bisphosphate). Arf6, a small GTPase switch protein, can stimulate AP2 and clathrin recruitment to the plasma membrane via activation of PIPKIγ, the major PIP2 generating enzyme (Krauss et al., 2003).

Clathrin is recruited to and assembles onto a network of endocytic accessory proteins bound to cargo molecules. During the initial stages of coated pit generation, the plasma membrane is still planar but coated with clathrin (Figure 4D).

During bud formation events, the plasma membrane acquires a positive curvature driven in part by the assembly of clathrin cages and adapter-mediated membrane bending (Ford et al., 2002). The mechanism by which planar clathrin-coated membranes are converted to coated buds is not very well understood, but may involve a change from a predominantly hexagonal organization of clathrin triskelia to

one that includes 12 pentagons in the final coat. Purified clathrin can also be induced to form cage-like structures in the absence of membranes (Fotin et al., 2004).

Fission of clathrin-coated vesicles is achieved by dynamin which acts as a mechanochemical enzyme that drives membrane vesiculation. Dynamin is also required for other endocytic processes like phagocytosis, caveolin-dependent endocytosis and raft- or microdomain-mediated endocytosis. During the budding step, dynamin molecules are recruited to the neck of a clathrin coated pit and encircle it as a molecular collar. GTP hydrolysis causes the constriction and helical expansion of this collar and leads to fission of the vesicle from the plasma membrane.

Figure 1.3 Sequence of events during clathrin mediated endocytosis. (A) Clathrin mediated endocytosis requires coated pit nucleation via selection of cargo proteins. A positive curvature is gradually introduced into the membrane to generate coated pits. GTP hydrolysis by dynamin supplies the energy for the fission event. Clathrin-coated vesicles are rapidly stripped off from their coats in the cytosol by the combined action of Hsc70 and auxilin (excerpted from Alberts et al., 2002, Molecular Biology of the Cell, 4^th Ed.). (B) One of the first electron microscope pictures showing clathrin-mediated endocytosis. Oocytes of the hen utilize CME in uptake of lipoprotein particles. The lipoprotein can be seen as a fuzzy layer on the extracellular facet of the plasma membrane (left side).

Note the CCVs generated as a result of this process are abnormally large in size compared to conventional CCVs. (Excerpted from Perry and Gilbert, 1979)

Uncoating of clathrin-coated vesicles is believed to be a rapid event compared to other steps. Therefore free clathrin-coated vesicles are rarely observed in electron micrographs of cells. The uncoating reaction involves the disassembly of the clathrin lattice encapsulating the transport vesicle. Hsc70, a 70kDa chaperone protein which belongs to the DnaK family of heat shock proteins, is targeted to clathrin-coated vesicles by auxilin. There it drives clathrin uncoating by providing energy via its ATPase function (Morgan et al., 2001; Takei and Haucke, 2001; Brodsky, 2001).

Synaptojanin 1, a phosphoinositide phosphatase at presynaptic nerve terminals, is another essential factor for uncoating of internalized synaptic vesicles. Its targeted deletion results in accumulation of coated vesicles near periactive zones (Cremona et al., 1999).