1. Conserved Structural Motifs in Intracellular Trafficking Pathways
Gregory R Hoffman, Peter B Rahl, Ruth N Collins, Richard A Cerione 
Molecular Cell 
Volume 12, Issue 3, Pages (September 2003)
DOI: /j.molcel

2. Figure 1
The Carboxyl-Terminal Region of γCOP Is an Evolutionarily Conserved Region Involved in the Normal Function of the COPI Complex
(A) The carboxyl-terminal region of the Saccharomyces cerevisiae γCOP homolog, Sec21p, is essential for viability. A haploid tester strain (RCY899), deleted for the genomic copy of SEC21 using the HIS5 module and carrying a wild-type copy of SEC21 on the pRS316 URA3 CEN plasmid, was transformed with various sec21 mutants carried on the pRS315 LEU2 CEN plasmid. Elimination of the wild-type gene was induced by selection on 5-FOA, and growth was assessed at 25°C and 37°C. The temperature-sensitive sec21-1, which contains a glycine to aspartic acid change at position 482, was used as a positive control when assaying for temperature-sensitive lethality and trafficking defects (Novick et al., 1980).
(B) The carboxyl-terminal region of Sec21p is involved in COPI-mediated trafficking. Tester strains expressing the indicated sec21 construct were selected on 5-FOA to ensure loss of the wild-type gene. These strains were grown at 25°C to an OD600 of 0.8 and shifted to 37°C for metabolic labeling experiments. In these experiments, the high molecular weight species corresponds to mature (glycosylated) invertase and is indicative of an intact secretory pathway, while the low molecular weight form corresponds to unprocessed invertase trapped in the ER due to a block in normal COPI-mediated trafficking events.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Structure of the Carboxyl-Terminal Domain of γCOP
(A) Ribbon diagram of the carboxyl-terminal domain of γCOP. An overall view of the γCOP appendage domain is shown. The immunoglobulin-like β sandwich is shown in yellow and the platform domain in green.
(B) Experimental electron density. A region of the solvent-flattened electron density map calculated for experimentally determined MAD phases is shown contoured at 1 σ in the vicinity of Met256. The position of the selenium atom in the Met256 residue is indicated by a peak in the isomorphous difference map contoured at 10 σ (red). The position of the final model refined against the 2.3 Å native data set is shown in a ball-and-stick representation.
(C) Comparison of the appendage domain of γCOP with that of α- and βAP2. The structures of the αAP2 appendage domain (left) and the βAP2 appendage domain (right) are shown oriented relative to the platform domain of γCOP.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Sequence Alignment of the Appendage Domains
A structure-based sequence alignment of the appendage domains of α- and βAP2 with that of γCOP was generated based on a structural alignment performed using the program O. Secondary structural elements of the γCOP and αAP2 appendage domains are indicated above the sequence alignment with the same color scheme as the structures in Figure 2. The blue arrow denotes the position of the domain boundary. Residues positioned on the basis of this structural alignment are indicated in black text. Strictly conserved residues in the structural alignment are highlighted in red, while regions of similarity, calculated using a Risler matrix, are boxed. Residues for which no structural information is available were positioned based on a primary sequence alignment using the ClustalW program and are indicated in gray text. The Sec21p sequence was positioned based on a primary sequence alignment with γCOP. The red circles indicate the position of the truncation mutants of γCOP and Sec21p discussed in the text, the first corresponding to γCOP:1-554 (Sec21:1-579) and the second corresponding to γCOP:1-617 (Sec21:1-676). The conserved F/W motif is indicated below the sequence alignment and is highlighted by an orange box, blue boxes highlight the positions of basic residues discussed in the text, and red boxes indicate residues elsewhere in the domain that are identical in all three structures. The sequence surrounding the F/W motif of βCOP is also included in the alignment, suggesting the presence of a putative appendage domain in the carboxyl-terminal domain of human βCOP.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Structural and Biochemical Similarities of the γCOP and AP2 Appendage Domains
(A) The F/W motif lies in a cleft on the surface of the platform domain. A view of the γCOP platform domain looking down the cleft formed between the α1 helix and the β sheet. The conserved phenylalanine and tryptophan side chains of the F/W motif are shown as a ball-and-stick representation. The β12-β13 loop of α- and βAP2 are shown in red and purple, respectively, demonstrating the more solvent-exposed nature of the F/W motif in the γCOP structure.
(B) Comparison of solvent-exposed residues surrounding the F/W motif in the platform domains. The surfaces of the platform domains of γCOP (green), αAP2 (red), and βAP2 (purple) are shown in a ribbon representation. The structures have been rotated 90° relative to their orientation in Figure 2, such that the top of the platform domain is facing the viewer. The conserved phenylalanine and tryptophan residues of the F/W motif found in all three platform domains are shown as a ball-and-stick model. In addition, critical residues near the F/W motif of α- and βAP2 involved in interactions with known binding partners are shown along with the residues at the corresponding positions in the γCOP platform domain.
(C) Comparison of the molecular surface of the appendage domains. The molecular surface of each appendage domain is shown colored on the basis of the electrostatic potential at the surface. Red indicates acidic regions and blue indicates basic regions. The appendage domains are shown in a similar orientation to Figure 2.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Functional Importance of the COPI Appendage Domains
(A) Appendage domain mutant analysis of Sec26p and Sec21p. The left panel shows the growth phenotype of haploid cells with mutations in the F/W motif of SEC21 and SEC26. RCY2590 cells, genotype MATα his3Δ0 leu2Δ0 ura3Δ0 lys2Δ0 SEC21ΔHIS SEC26ΔKANR [pRS316 SEC21 SEC26], were simultaneously transformed with pRS315 sec21 plasmids and pRS317 sec26 plasmids as indicated. Transformants were spot diluted onto 5-FOA to eliminate the wild-type SEC21 and SEC26 pRS316 plasmid, in order to assess growth with the mutant versions of the genes as the sole copy. No effect could be observed for mutation of the phenylalanine residues of SEC21 that correspond to the F/W motif, either alone or in combination with a mutant version of the SEC26 F/W motif. Mutation of the SEC26 F/W motif alone gave rise to a temperature-sensitive phenotype at 40°C, indicating a requirement for the F/W motif for the full functionality of this protein in vivo. The right panel shows the effects of single and double appendage domain mutations of SEC21 and SEC26. The F/W mutation of SEC26 is synthetically lethal in combination with removal of the appendage domain from SEC21 (this corresponds to Sec21:1-676 in Figure 1) in comparison to either mutation alone.
(B) Specificity of appendage domain ligand interactions. GST-fusion proteins (25 μg, 100 μg, and 250 μg) encoding the appendage domains for γCOP (residues 555–874), αAP2 (residues 701–938), and βAP2 (residues 700–937) were incubated with rat brain cytosol (2 mg of total protein). The GST-appendage domains were precipitated with glutathione-agarose beads, and associated proteins were detected by Western blot analysis using the indicated antibodies.
(C) Bound sulfates in the γCOP platform domain occupy a position similar to that of the FxDxF peptide ligand in the αAP2 appendage domain. A pair of sulfate ions (shown in magenta) occupies the putative ligand binding site in the platform domain of γCOP. The hydrogen bonding interactions involved in stabilizing these sulfate ions are shown as compared to hydrogen bonding interactions between residues at corresponding positions in the αAP2 platform domain and the FxDxF peptide from amphiphysin (shown in magenta).
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Model for COPI Assembly
(A) A proposed model of the conserved structural features of COPI and clathrin coated vesicle formation. A model of the AP2 complex and its interaction with clathrin is shown, similar to that presented in Bonifacino and Lippincott-Schwartz (2003). A proposed model for the COPI complex, based on the conserved structural features with the AP2 complex and clathrin described in the text, is shown above the AP2 model.
(B) Coimmunoprecipitation of COPI subunits in mammalian cells. HA-tagged constructs of γCOP were transfected into COS7 cells, immunoprecipitated from cell lysates, and subjected to Western blot analysis using an antibody against βCOP. The lower panel shows the amount of each γCOP construct in the precipitates.
Molecular Cell  , DOI: ( /j.molcel )