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1.2.1 Clathrin-mediated endocytosis

Eukaryotic cells use endocytosis for a variety of purposes, including nutrient uptake, SSV recycling, remodeling of the plasma membrane, regulation of cell-surface expression of signalling receptors, and the establishment of cellular polarity. It is therefore not surprising that cells employ differently regulated endocytic pathways, according to the cargoes and their final destinations (Figure 1.10). Clathrin-mediated endocytosis is so far the best-characterised endocytic process (Marsh and McMahon, 1999). However, recent work has highlighted the existence of other types of endocytic pathways, such as caveolar uptake, various clathrin-independent mechanisms and macropinocytosis, which still remain largely unknown (Nichols and Lippincott-Schwartz, 2001).

Clathrin-mediated endocytosis is used to internalise a variety of ligands, such as growth- factors, antigens and recycling receptors and is involved in two crucial transport steps:

endocytosis from the plasma membrane to early endosomes and transport from the trans-

Golgi network (TON) to endosomes. In addition, it can also function in budding from endosomes, immature secretory granules and other sites in the cell (Kirchhausen, 2000; Mellman, 1996).

In neurons the best-studied example of clathrin-mediated endocytosis is recycling of SSVs after exocytosis (Murthy and Stevens, 1998; Slepnev and De Camilli, 2000). The process is characterised by the progressive assembly of a clathrin scaffold which provides a mechanical means to deform the membrane into a “coated pit” (Marsh and McMahon, 1999) (Figure 1.11). A similar mechanism is also used for transferrin internalisation which is confined to the somatodendritic domain (Mundigl et al., 1993; Parton et al., 1992). In this first phase, cargoes and cargo receptors are concentrated by a still poorly understood

Clathrin- d ep en d en t 2 CLav' -oiwLi- p-nr 0 't 3 Clathrin- Dynamin ndependent Dynamin 4 Macro­ pinocytosis PI3Kinase lactin

Figure 1.10 Schematic representation of structures involved in endocytic pathways.

The "classical" coated pathway (1) requires the participation of the clathrin coat and the dynamin-dependent fission of the budding endocytic vesicle. However, endocytosis can occur also through caveolae, uncoated flask-shaped invaginations characterised by the presence of caveolins (2). These endocytic structures may also require dynamin for the fission step. Clathrin- and caveolae-independent pathways have also started to emerge (3). They may possibly involve the participation of lipid rafts, but the molecular components involved are still unknown. Macropinocytosis (4) consists of fluid internalisation in large endocytic vesicles formed after the closure of lamellipodia.

The process involves the participation of the actin cytoskeleton and PI3 kinase. (Adapted from Sandvig, EMBO .1, 2()00).

mechanism into the budding vesicle, which then matures and eventually pinches off from the plasma membrane. A subsequent energy-dependent uncoating step follows, giving rise to a vesicle ready to continue its trafficking in the cytoplasm or, in the case of a SSV, to be refilled with neurotransmitter and re-enter the exocytic cycle (Brodin et al., 2000).

The unit elements of the clathrin lattice are three-legged triskelia, comprising three heavy and three light chains that form stable oligomeric complexes (Figure 1.12). The clathrin coat is assembled on the cytoplasmic face of the plasma membrane by the recruitment of the adaptor complex AP-2, a heterotetramer consisted of two large subunits, a and P2 (each of ~ 100 kDa), and two smaller chains, \i2 (50 kDa) and a2 (20 kDa). The AP-2 complex not only links clathrin to the membrane, but also coordinates coat assembly with the selection of protein or lipid cargoes, concentrating them in the emerging bud (Kirchhausen, 2000). Indeed, the AP-2 core region binds both to membrane proteins, such as synaptotagmin and proteins containing endocytic motifs, and to membrane lipids, such as phosphoinositides (Pis) (Bonifacino and Dell'Angelica, 1999; Gaidarov and Keen, 1999; Slepnev and De Camilli, 2000; von Poser et al., 2000). The binding of AP-2 to clathrin is instead mediated primarily by the P2 subunit at the level of the “ear” domain and in the hinge region connecting the ear to the trunk domain (Robinson and Bonifacino, 2001; Shih et al., 1995). The coat also contains an additional protein named A PI80 in neurons and CALM in other cells which is able to interact with clathrin, the AP-2 ear domain and membrane Pis, thereby assisting coat assembly and possibly controlling vesicle size (Ford et al., 2001; Tebar et al., 1999; Zhang et al., 1998). A plethora of accessory factors assisting the formation of clathrin-coated vesicles have been identified (Slepnev and De Camilli, 2000; Takei and Haucke, 2001) (Figure 1.12). These factors help coordinate coat formation with biochemical changes in the lipid bilayer and local

Clathrin Uncoating PIP2 AP-2 Cargo Dynamin A P180 Actin Synaptotagm in Fission %

Coat nucléation and assem b ly Maturation

Figure 1.11 Schematic representation of clathrin-dependent endocytosis of SSV.

The AP-2 adaptor protein acts as a bridge between membrane proteins and lipids and the clathrin coat, which enables the formation of a "coated pit" enriched in lipid or protein cargoes. The process can be assisted by API80. The actin cytoskeleton and a vast array of accessory factors is involved in the maturation of the emerging bud, which udergoes a dynamin-dependent fission step. The final uncoating process requires synaptojanin-dependent PtdIns(4,5)P2 (PIP^) hydrolysis and other accessory factors such as hsc70 and auxilin.

AP-2 API 80/ CALM M em brane f a c to rs Synaptotagm in A c c e sso ry p ro te in s Clathrin asse m b ly Dynamin ('o T P a s e ^ PH Endophilin C lathrin/ AP-2 binding SH3 Amphiphysin Intersectin Epsin Synaptojanin Auxilin S H 3 S H 3 PH S H 3 SH 3 SH3 m NPF ( ENTH ] DPW ÿ B 5' P h o s p h a ta s e C lathrin/ AP-2 binding Stoned B/ Stonin 2 hsc70 Œ) |i2 N PF Hom ology SBD [ T P a se| I I

Heterotetrameric com plex o f a (100-110 kDa), p2 (105 kDa), (50 kDa) and o 2 (20 kDa) subunits that links the clathrin shell to the membrane through interactions o f its p2 and a subunits with membrane proteins and lipids.

Accessory component (95-kDa protein; runs at 180- kDa on SDS-PAG E) o f clathrin coats that might regulate vesicle size. Contains PlP^-binding ENTH and clathrin-assembly domains.

AP-2 binding protein o f synaptic vesicles and the presynaptic plasmalemma that facilitates vesicle recycling by promoting coated pit nucléation. 100-kDa GTPase that polymerises into oligom eric rings at the neck o f invaginating buds and catalyses vesicle fission upon G IT hydrolysis.

Lysophosphatidic acid acyl transferase (4 0 kDa) with putative roles in coated bud maturation and vesicle fission. Binds to dynamin and synaptojanin. Binding partner o f clathrin, AP-2 and dynamin. Putative role in fission.

Large modular protein (145-200 kDa isoforms) with multiple partners in endocytosis.

fw o related proteins o f 90-94 kDa that bind to clathrin, AP-2, Eps 15 and PIP^.

Inositol-phosphatase (145-170 kDa isoforms) that regulates PIP^ metabolism and the stability o f clathrin-AP-2 coats. 'ITie 170-kDa isoform contains NPF motifs.

J-domain protein (100 kDa) that assists hsc70 in