Cargo Sorting at the trans-Golgi Network for Shunting into Specific Transport Routes: Role of Arf Small G Proteins and Adaptor Complexes

Abstract

The trans-Golgi network (TGN) is responsible for selectively recruiting newly synthesized cargo into transport carriers for delivery to their appropriate destination. In addition, the TGN is responsible for receiving and recycling cargo from endosomes. The membrane organization of the TGN facilitates the sorting of cargoes into distinct populations of transport vesicles. There have been significant advances in defining the molecular mechanism involved in the recognition of membrane cargoes for recruitment into different populations of transport carriers. This machinery includes cargo adaptors of the adaptor protein (AP) complex family, and monomeric Golgi-localized γ ear-containing Arf-binding protein (GGA) family, small G proteins, coat proteins, as well as accessory factors to promote budding and fission of transport vesicles. Here, we review this literature with a particular focus on the transport pathway(s) mediated by the individual cargo adaptors and the cargo motifs recognized by these adaptors. Defects in these cargo adaptors lead to a wide variety of diseases.

Keywords: AP-1; AP-3; AP-4; GGAs; adaptor proteins; post-Golgi transport; protein sorting; trans-Golgi network.

Figure 1

Membrane trafficking pathways at the trans-Golgi network (TGN): (A) a schematic showing the post-Golgi trafficking pathways (red arrows) and the retrograde recycling pathways (black arrows) from the plasma membrane and endosomal/lysosomal compartments back to the TGN; (B) the distinct trafficking pathways at the TGN are regulated by specific cargo adaptor proteins including adaptor protein (AP) complexes (AP-1 in red, AP-3 in blue, AP-4 in orange); and (C) monomeric Golgi-localized, γ ear-containing, Arf-binding proteins (GGA1-3 in brown).

Figure 2

Cargo sorting at the TGN: (A) schematic showing the process of membrane recruitment of the adaptor protein complex, AP-1, and the binding of sorting motifs on the cytoplasmic tail of cargoes. GTP-bound membrane-associated small G protein Arf1 recruits cytosolic AP-1 to the TGN membranes. Arf1 also induces conformational change in AP-1, from closed to an open conformation, to allow the binding of cargo sorting motif(s). The μ1 subunit (green) binds tyrosine-based sorting motif (YXXΦ, green cargo) and the σ1−γ subunits bind dileucine sorting motif (DE)XXXL(LI), brown cargo). (B) Schematic representation of the membrane recruitment of AP complexes by active small G proteins, followed by binding of AP complex to the cytoplasmic tail of cargo proteins. Subsequently, the hinge-ear extensions of each AP complex mediate the recruitment of coat and accessory proteins to drive membrane curvature and vesicle formation.

Figure 3

Regulation of Arf family small G proteins: (A) The state of the Arf small G proteins GTP binding and hydrolysis is regulated by Arf family guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), respectively. The membrane-associated GTP-Arf then recruits effectors such as cargo adaptor proteins, membrane lipid-modifying enzymes, and additional GEFs. (B) GDP-Arf associates with the surface of the membrane in a reversible manner. Exchange of GDP for GTP in Arf causes a conformational change of the switch (SW1 and SW2) and interswitch regions to enter the hydrophobic pocket occupied by the myristoylated N-terminal helix (red). GTP-Arf then associates tightly with the membrane via the exposed N-terminal helix.

Figure 4

Cargo adaptor proteins: Five adaptor protein (AP) complexes, AP-1 (red), AP-2 (green), AP-3 (blue), AP-4 (orange), and AP-5 (pink), have been identified to date in higher eukaryotes. Each heterotetrameric AP complex comprises two ~100 kDa large subunits (β1–5, and either α, γ, δ, ε, or ζ), one ~50 kDa medium subunit (μ1–5), and one ~20 kDa small subunit (σ1–5). Together, they form the AP core, which is important for membrane recruitment and cargo sorting motif recognition. The C-termini of both large subunits of each AP complex give rise to the hinge and ear domains for further recruitment of accessory proteins. AP-1 is localized at the TGN/recycling endosomes and regulates bidirectional transport. AP-1 also regulates basolateral sorting in polarized cells. AP-2 is responsible for endocytosis of cargoes from the cell surface. AP-3 is localized at the TGN/early endosomes and regulates transport to the late endosomes/lysosomes. AP-4 is localized at the TGN and regulates cargo trafficking from the TGN to the early endosomes. AP-5 is localized at the late endosomes/lysosomes, and the trafficking pathway it regulates is still unclear. The three Golgi-localized γ-ear containing Arf binding isoforms, GGA1, GGA2, and GGA3 (brown), are monomeric and have similar structural protein folding to the ear domain of AP-1γ subunit. GGAs are localized at the TGN and endosomes.

Figure 5

Tyrosine sorting motifs binding sites on AP complex. Crystal structures of the C-terminal domains of μ subunits of AP-1 through 4 in association with tyrosine-based sorting motifs (underlined). The C-terminal domains of μ1–4 are further subdivided into subdomain A (blue) and subdomain B (red). (A) Mouse AP-1 μ1 C-terminal domain in complex with class 1 major histocompatibility complex (MHC-1) peptide SYSQAA (PDB: 4EN2), (B) rat AP-2 μ2 C-terminal domain in complex with epidermal growth factor receptor (EGFR) peptide FYRAL (PDB: 1BW8), (C) rat AP-3 μ3A C-terminal domain in complex with TGN38 peptide SDYQRL (PDB: 4IKN), (D) human AP-4 μ4 C-terminal domain in complex with amyloid precursor protein (APP) peptide TYKFFEQ (PDB: 3L81).