Table 2: Accessory proteins involved in clathrin and COP coat formation.

 

AP1/GGA-associated proteins

AP2-associated proteins

COPI-associated proteins

COPII-associated proteins

Accessory factors involved in clathrin-AP mediated trafficking events.

Accessory factor

Salient features

Peptide motifs

Functions

Amphiphysin

All forms have an N-terminal BAR domain (a dimerisation, lipid binding and curvature sensing module)[1].

All mammalian forms have a C-terminal SH3 domain that binds dynamin[2].

The non-muscle splice forms have an insert that binds clathrin and the AP2 complex.

AP2 a-adaptin appendage binding motifs: FxDxF and DxF[3].

Clathrin b-propeller binding motifs: LLDLD and PWxxW[4].

SH3 domain binds to PSRPNR motif in dynamin[2].

Induces/senses membrane curvature.

Recruits dynamin to membranes. Promotes clathrin polymerisation on membranes.

 

AP180/CALM

N-terminal ANTH domain binds PtdIns(4,5)P2

C-terminal region binds clathrin and the AP2 complex[5, 6].

AP2 a-adaptin appendage binding motifs: FxDxF and DxF motifs.

CALM has DxF and NPF motifs (bind to EH domains).

Clathrin binding motifs are not well defined.

Recruits clathrin and promotes polymerization on membranes containing PtdIns(4,5)P2. Regulates vesicle size.

ARFGAP

GTPase activating protein for ARF[7]. Binds betaWD40 and gamma-COP appendage [8].

The peptide motif in ARFGAP has not been described but is likely to resemble that found alpha and gamma appendage binding partners.

Activates ArfGTP hydrolysis to promote coat disassemby

ARFGEF

GEF for Arf

 

Converts ArfGDP to ArfGTP.

ARF1

ARF1

Small G-protein

GTP binding motifs G1 to G4: G1(GKT); G2(T); G3(VGG); G4(NKQD).

Adaptor complexes and COPI subunits are effector molecules[9].

Helps to recruit AP1, AP3, GGA and COPI F-subcomplex to membranes in a GTP dependent manner.

ARH1/2

N-terminal PTB domain binds to inositol lipids and receptor tails[10]. Binds to NPXY motifs and AP2[11]

PTB domain recognizes NPVY motif in LDLR.

Clathrin b-propeller binding motif: LLDLE:

Also binds b2-adaptin (motif not defined)[12].

An alternative cargo adaptor. May also be found in combination with other adaptors.

b-arrestin

Similar to visual arrestin whose structure has been solved. This domain binds inositol lipids and G-protein coupled receptors. A C-terminal extension binds clathrin and the AP2 complex.

Clathrin b-propeller binding motif: LIEFE[13].

AP2 b2-adaptin appendage binding motif: IVFEDAFR with the arginine being essential and corresponding to R-394 in b2-arrestin [13].

An alternative cargo adaptor that helps recruit clathrin and AP2 in G-protein coupled receptor endocytosis.

Auxilin

Binds clathrin and contains a DnaJ domain and is a cofactor for Hsc70.

AP2 a-adaptin appendage binding motifs: DPW and WDW.

Clathrin b-propeller binding motifs: DxF and

DLL.

DnaJ domain has a HPDK signature motif conserved across all auxilins and is required for Hsc70 binding and stimulation of its ATPase activity.

Involved in uncoating of clathrin-coated vesicles.

Dab2/Doc2

N-terminal PTB domain binds to PtdIns(4,5)P2 and receptor tails [14]. NPXY motifs and AP2 [15]

PTB domain recognizes FxNPxY motif in the LDL receptor.

AP2 appendage binding motifs: FxDxF and DxF[16].

An independent cargo-specific adaptor that also binds to the AP2 complex.

Dsl1p

Interacts with alpha and delta COP and ER resident protein Tip20p[17]

Contains WxW and WxxxW motifs in an acidic central domain

Integrates cargo with the ?clathrin-? and ?AP-like? subcomplexes of COPI

Dynamins

Large GTPase that self-oligomerises into a helix on negatively charged membranes. Changes conformation on GTP hydrolysis.

GTP binding motifs G1 to G4: G1(GKS); G2(T); G3(DLPG); G4(TKLD).

Vesicle scission on GTP hydrolysis.

Endophilins

N-terminal BAR domain (a dimerisation, lipid binding and curvature sensing module) [18]. C-terminal SH3 domain that binds synaptojanin and dynamin[19].

SH3 domain binds to PPxRP motif in dynamin and PxRPP motif in synaptojanin [20, 21].

Induces/senses membrane curvature.

Recruits the lipid phosphatase, synaptojanin, to membranes, and thus is involved in uncoating.

Epsin1

EpsinR

N-terminal ENTH domain of epsin1/epsinR binds PtdIns(4,5)P2 and PtdInsP respectively and promotes initial membrane curvature[22, 23]. Epsin1 has 3 ubiquitin binding motifs (UIMs)[24, 25]. Both epsin1 and epsinR have a clathrin/adaptor binding domain.

AP2 a-adaptin appendage binding motifs in epsin1: DPW.

AP1 g-adaptin appendage binding motifs in epsinR: DFxDF.

Clathrin b-propeller binding motif in epsin1 is LMDLADV and LVDLD, and is DLFDLM in epsinR[23, 26].

Eps15 EH domain binding motifs: 3 copies of NPF motif in epsin1[27].

The ubiquitin binding signiture is ExxxLxLAxAxS[K/R/Q][24].

Induces membrane curvature in conjunction with promoting clathrin polymerization. Epsin1 may bind ubquitinated cargo via its UIMs.

Eps15

Eps15

N-terminus has 3 EH domains that bind the NPF motifs in epsin1[28]. Also has a clathrin/adaptor binding domain[29].

AP2 a-adaptin and b-adaptin appendage binding motifs in epsin1: multiple DxFs

Located at the edges of clathrin-coated pits[30]. Thought to organize epsins and other molecules at the leading edge of coated pit formation.

HIP1/HIP1R

N-terminal ANTH domain binds PtdIns(4,5)P2. C-terminal region binds clathrin, the AP2 complex and actin [31-34]

AP2 a-adaptin appendage binding motifs in HIP1: FxDxF and DxF [3].

Clathrin b-propeller binding motif in HIP1: LMDMD.

[35]

Clathrin b-propeller binding motif in HIP1R: LIEIS.

Thought to provide a link between actin and clathrin nucleation.

Intersectin/DAP160

Has multiple EH and SH3 domains and a RhoGEF domain. Binds N-WASP, cdc42, dynamin and SNAP-25 among other proteins.

Binds proline rich motifs in ligands through its SH3 domains and NPF motifs in ligands via its EH domains.

Links actin polymerisation to proteins involved in clathrin-coated vesicle formation.

P56

Binds to g-adaptin and GGA appendage domains in vitro and co-localises with GGAs in vivo [36, 37].

GGA/AP1 g-adaptin appendage binding motifs: DFxxF.

Unknown function

PACS1

Binds to AP1 and AP3 complexes and acidic cluster motifs in cargo such as M6PR, Nef and furin [38, 39]

AP1 complex interaction via ETELQLTF sequence (with mu and sigma subunits)[39].

 

Recruits cargo containing diacidic motifs.

Sar1p

Small G-protein[40].

GTP binding motifs G1 to G4: G1(GKT); G2(T); G3(DLGG); G4(NKID).

Binds to membranes when occupied by GTP and recruits COPII coat components[41].

GTPase activity implicated in coat disassembly upon vesicle budding.

Stonins (1/2)

Eps15, AP2 and synaptotagmin[42, 43]

AP2 a-adaptin appendage binding motif: WVxF[44].

Eps15 EH domain binding motifs: 2 copies of NPF in stonin2.

 

Assists AP2/synaptotagmin recruitment

Synaptojanin

Phosphoinostide 5?-phosphatase and SacI phosphatase domain[45, 46]. Binds to the AP2 complex and has a proline rich domain that binds to endophilin and amphiphysin[21, 47].

AP2 a-adaptin appendage binding motifs: FxDxF and WxxF are found in the 170kDa splice form and may be involved in the recruitment of synaptojanin to coated pits[3, 43].

A lipid phosphatase that is recruited to coated vesicle via endophilin. By dephosphorylation of inositol lipids this will weaken the attachment of coat proteins and thus this protein is likely involved in uncoating.

Synaptotagmin1

Calcium sensor for exocytosis. Gets endocytosed by binding to the AP2 complex and to stonin2[42, 48-52].

WHxL motif is essential for plasma membrane association[53].

 

May integrate exo-and endocytosis in nerve terminals

Syndapin/Pacsin

C-terminal SH3 domain. Binds N-WASP and dynamin [54, 55].

 

Regulates clathrin-coated vesicle motility

Hsc70

ATPase that binds to auxilin [56, 57]

 

Clathrin coat release. Hsc70 is also called the ?uncoating ATPase?.

g-synergin

Has EH domain that binds to NPF motifs in SCAMP[58]. Also binds to the AP1 complex[59].

AP1 g-adaptin appendage binding motif: DFxDF[23].

Unknown function in clathrin-AP1 trafficking.

 

 

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