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Review
, 21 (3), 159-67

Transport According to GARP: Receiving Retrograde Cargo at the trans-Golgi Network

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Review

Transport According to GARP: Receiving Retrograde Cargo at the trans-Golgi Network

Juan S Bonifacino et al. Trends Cell Biol.

Abstract

Tethering factors are large protein complexes that capture transport vesicles and enable their fusion with acceptor organelles at different stages of the endomembrane system. Recent studies have shed new light on the structure and function of a heterotetrameric tethering factor named Golgi-associated retrograde protein (GARP), which promotes fusion of endosome-derived, retrograde transport carriers to the trans-Golgi network (TGN). X-ray crystallography of the Vps53 and Vps54 subunits of GARP has revealed that this complex is structurally related to other tethering factors such as the exocyst, the conserved oligomeric Golgi (COG) and Dsl1 (dependence on SLY1-20) complexes, indicating that they all might work by a similar mechanism. Loss of GARP function compromises the growth, fertility and/or viability of the defective organisms, emphasizing the essential nature of GARP-mediated retrograde transport.

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure I
Figure I
Figure 1
Figure 1
Proposed role of GARP in tethering retrograde transport carriers to the trans-Golgi network. (a) Protein cycling between the TGN and endosomes. Some transmembrane proteins, including acid hydrolase receptors, processing proteases and SNAREs, cycle between the TGN and endosomes [86,87]. Retrograde transport of these proteins from endosomes to the TGN occurs through a tubular compartment referred to as the tubular endosomal network (TEN). From this compartment, tubular or vesicular transport carriers (TCs) deliver cargos to the TGN [86]. (b) Schematic representation of tethering mediated by GARP. GARP is shown as a heterotetramer assembled through the amino-terminal regions of its four subunits [13,15,17,18,22]. GARP has been shown to interact with small GTPases of the Rab and Arl families (Ypt6 and Arl1, respectively in S. cerevisiae), which might contribute to GARP recruitment to the TGN [11,35]. GARP also interacts with the Habc domain of a t-SNARE (Tlg1 in S. cerevisiae and Syntaxin 6 in humans) [11,15,14,18,41], probably leading to displacement of this domain from the SNARE domain and thus enabling pairing with other SNAREs. Weaker interactions with other SNAREs [12,22] might further promote SNARE complex formation. The specifics of this graphic representation are highly speculative, since the molecular details of the interactions have not been worked out.
Figure 2
Figure 2
GARP localizes to the TGN, where it enables cargo transport from endosomes. (a–c) Co-localization of GARP (labeled by expression of a Vps54-GFP chimera) (green channel) with the TGN marker TGN46 (labeled with a specific antibody to this protein followed by Alexa-594 conjugated secondary antibody) (red channel) in human HeLa cells imaged by confocal fluorescence microscopy. Panel c shows a merged image in which yellow indicates co-localization. The TGN appears as a cisternal/tubular network partially surrounding the nucleus (Nu). Cy: cytoplasm. (d,e) Evidence for the involvement of GARP in retrograde transport. Confocal fluorescence microscopy shows that internalized Cy3-conjugated B subunit of Shiga toxin (STxB) reaches the TGN in control HeLa cells, but accumulates in endosomes and retrograde transport carriers in HeLa cells depleted of Vps51 (i.e., Ang2) by RNAi knock-down (KD). Images are reproduced from refs. [13] (a–c) and [18] (d,e).
Figure 3
Figure 3
Characteristics of the GARP subunits. (a) Schematic representation of the human GARP subunits. N and C represent the amino- and carboxy-termini of the proteins. The scheme shows the approximate sizes of the four subunits and the location of predicted coiled-coil (CC) motifs. The presence of coiled coils, particularly in the amino-terminal regions, is a conserved feature of GARP subunits from all species. Analyses using the SMART server (http://smart.embl-heidelberg.de/) predict the presence of a C2H2-type zinc finger at the amino-terminus of Vps54 from Drosophila and C. elegans but not from other species. These domains generally function as binding sites for other macromolecules, but their exact role in these Vps54 orthologs is unknown. (b) Crystal structures of carboxy-terminal (CT) fragments from S. cerevisiae Vps53 [25] and human Vps54 [24] in comparison to those of a carboxy-terminal (CT) fragment from the S. cerevisiae Sec6 subunit of the exocyst complex [30] and the full-length S. cerevisiae Tip20 subunit of the Dsl1 complex [27]. Structures are represented as ribbon diagrams with the tandem α-helical-bundle domains (designated A–E) shown in different colors.
Figure 4
Figure 4
Functional regions of GARP subunits. (a) Ribbon diagrams of S. cerevisiae Vps53 and human Vps54 carboxy-terminal (CT) fragments shown under a translucent surface. Segments corresponding to the different α-helical-bundle domains are indicated in green (C domain), blue (D domain) and red (E domain). The yellow highlight on Vps53-CT indicates a cluster of highly conserved residues that are required for pro-CPY sorting to the vacuole [25]. The magenta highlight on Vps54-CT indicates a hydrophobic pocket containing the leucine-967 residue (red stick model) that is mutated in the wobbler mouse [24]. (b) Defective motor function of the Vps54-mutant wobbler (wr) mouse. Unlike a normal mouse (+/+), a homozygous wobbler mouse (wr/wr) cannot grab onto the grid with either the forelimbs or hindlimbs (arrows) as a result of motor neuron degeneration. Insertion of a normal Vps54 allele (wr/wr-Vps54-tg) into the genome rescues the phenotype of the mutant mouse. Photograph courtesy of Thomas Schmitt-John (Aarhus University, Denmark). Reproduced with permission from ref. [61].

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