The GAT Domain

C-terminal domain of the Vps27 family SH3, Src-homology 3 domain gEAR, y-adaptin ear domain. The UIM motifs are shown as small black ellipses, the CAT domains as larger gray ones. 

The GGA proteins (three in mammals, two in yeast) also contain a so-called y-adaptin ear domain at their C-terminus, which is lacking in the three mammalian Tom 1-like proteins. 

Initially, the role of the GAT domain in GGA proteins was seen in the binding of small GTPases of the Arf family, a critical step in the recruitment of clathrin to the TGN membrane [92]. However, the GAT domains of the Toml-like family do not bind to Arf. Recently, GAT domains of both protein classes were found to bind to ubiquitin and it was possible to separate the two binding sites to different subdomains of the GAT domain [91]. A number of X-ray structures of GAT domains are available [93-95], presenting the domain as an elongated three-helix bundle. Unlike the UBA-like structures, the GAT helices are almost parallel and considerably longer. As a prominent feature, the N-terminal helix is much longer than the others this N-terminal extension contains the Arf interaction site and is not conserved in the Toml-family [93]. 

---

The GAT domain (GGA and Toml) has recently joined the ranks of ubiquitin-binding domains [91]. As the name implies, this domain is found in the GGA-and Toml-like proteins, two regulator classes of clathrin-mediated vesicular traffic. All proteins harboring the GAT domain also contain an N-terminal VHS domain, which is named after the Vps27, Hrs, and STAM proteins. Interestingly, these latter proteins are known to contain a ubiquitin-binding UIM motif, which appears to be replaced by the GAT domain in the GGA and Toml-like proteins (Figure 12.6). 

SuEE, S., Misea, S., Saidi, L. F., and Hurley, J. H., Structure of the GAT domain of human GGAl a syntaxin amino-terminal domain fold in an endosomal trafScking adaptor, Proc. Natl. Acad. Sci. USA, 2003, 100, 4451. 

For the assays described in this chapter, constructs comprised of the GAT domain of GGA proteins, and additional domains as necessary for the question being addressed, are expressed in bacteria. The proteins are... 

Fig. 1. Two-hybrid analyses of interactions between Arfs and the GAT domains. (A) Schematic representation of the domain organization of human GGAl and alignment of the GAT domain sequences of human (h) and S. cerevisiae (sc) GGAs. Residues conserved in all GGAs are shown as white letters on a black background, and those conserved in at least three members are shaded in gray. Positions of mutated residues identified by the reverse two-hybrid screening are indicated. (B) Growth of yeast cells harboring indicated combinations of a bait vector for Arf and a prey vector for the GAT domain on plates lacking tryptophan, leucine, and histidine. Reprinted from Biochemical Journal (Takatsu et al, 2002) with the permission of The Biochemical Society.

The GAT domain of each GGA protein fused to the C-terminus of GST is expressed in E. coli BL21(DE3) cells and purified using glutathione-Sepharose 4B beads (Amersham Biosciences) as described in the manufacturer s instruction. The buffers used are as follows GST-fusion protein binding buffer PBS, pH 7.4,5 mM /3-mercaptoethanol, 5 mg/ml DNase I, 5 mg/ml RNase A, and a protease inhibitor mixture (Complete -EDTA free, Roche Diagnostics, Indianapolis, IN) GST-fusion protein washing buffer PBS, pH 7.4, 5 mM /3-mercaptoethanol, Complete -EDTA free. 

The two-hybrid data were confirmed by pull-down assays. As shown in Fig. 2A, upper panel, the GST-fusion proteins of the GAT domains of all GGAs efficiently pulled down wild-type Arfl in the presence of GTP7S, but with very low efficiency in the presence of GDP. In contrast to wild-type Arfl, the pull-down efficiencies of Arfl(Q71L) were much higher,... 

These data obtained by two-hybrid and pull-down experiments consistently indicate that the GAT domains from all GGAs interact with GTP-bound Arfs, and that the GAT domain interacts with all classes of Arfs with comparable efficiencies. 

For expression of N-terminally HA-tagged GGA and the GAT domain, the cDNA fragments were separately subcloned into the pcDNA3-HAN vector (Shin et al, 1997). An expression vector for C-terminally Myc-tagged Arfl(Q71L) was described previously (Takatsu et al, 2002). 

Arfs have been shown to be responsible for membrane recruitment of coat protein complexes, including the COPI and AP-1 complexes (Nie et al, 2003 Shin and Nakayama, 2004). To examine whether interaction of Arf and the GAT domain is required for recruitment of GGAs onto TGN membranes, we performed the following experiments. When... 

---