Figure 2.

GGA GAT binding to PI4P. (A) GGA domain organization and structure. Left, the modular organization of the GGA domains. The GGA1 domain boundaries are as defined by Suer et al. (2003). The GAT domain is further divided into the N-GAT and C-GAT domains, which bind Arf1 and Ub, respectively. Our data showed that C-GAT also binds PI4P. Middle, a ribbon diagram of the X-ray crystal structure of GGA1 GAT, adapted from Collins et al. (2003). The GAT domain has four helices (named α1–4). The N-GAT domain has a hook-helix (α1 and part of α2), and the C-GAT domain has a three-helix bundle that is comprised of a portion of α2 and the entire α3 and α4. The PI4P-binding residues, GGA1 R260 and R265 (equivalent to GGA2 R276 and R281) are highlighted in red. We showed the GGA1 GAT structure instead of the GGA2 GAT structure here because only the former has been solved at the atomic level. Right, an expanded view of the GGA1 C-GAT structure. The C-GAT domain is rotated slightly in order to display the relation between the Ub-binding sites and PI4P-binding residues. Red, PI4P-binding residues; blue and green, Ub-binding residues in sites 1 and 2, as defined in yeast (Bilodeau et al., 2004). Additional site 1 residues identified by X-ray crystallography of the mammalian GGA GAT:Ub (Bilodeau et al., 2004; Kawasaki et al., 2005; Prag et al., 2005) that fall outside of these regions are not highlighted to simplify the presentation (see panel B). The site 1 residues were observed to bind Ub in the GGA3 GAT:Ub structure by Prag et al. (2005) and Kawasaki et al. (2005). The site 2 binding residues were identified by site-directed mutagenesis (Bilodeau et al., 2004; Puertollano and Bonifacino, 2004; Shiba et al., 2004). (B) Sequence alignment of the human and yeast GAT domains. Rectangles, helices in GAT; dotted lines, linkers between helices. The boundaries used to generate the N-GAT and C-GAT are indicated. Red letters denote basic amino acids that were conserved among different species. GGA1 R260 and R265 are conserved in mammalian and yeast GGAs, except that there is a two-residue offset in the yeast proteins. Red arrowheads, PI4P binding residues identified in this study. Blue lines, Ub-binding sites 1 and 2 that were identified in yeast (Bilodeau et al., 2004). Red lines, site 1 residues observed to bind Ub in GGA3 GAT:Ub crystals (Kawasaki et al., 2005; Prag et al., 2005). There is currently no crystal structure of GAT site 2 bound to Ub. The mammalian site 2 residues identified by site-directed mutagenesis (Bilodeau et al., 2004; Puertollano and Bonifacino, 2004; Shiba et al., 2004) are not highlighted here. (C) Comparing the binding of GGA2 domains to liposomes. 0.02 mg/ml purified GGA2 GST-VHS-GAT, GAT, and VHS were incubated with 0.2 mg/ml mixed lipid vesicles containing 15% PS or PI4P. The liposomes were collected by centrifugation and the bound GGA proteins were detected by Coomassie blue staining. The bottom panel shows the ratios of protein cosedimentated with PI4P versus PS liposomes (PI4P-binding index; mean ± SE, n = 5 or 6). (D) Comparing the binding of GGA1 and GGA2 GAT to PI4P-containing liposomes. GAT proteins (0.02 mg/ml) were incubated with increasing amount of liposomes. (E) Comparing the binding of GGA2 VHS and GAT domains to lipid dots. Proteins were incubated with PIP-arrays (Echelon, dotted with lipids ranging from 100 to 1.6 pmol), and the bound GGA domains were detected with anti-GST antibody.