The Sec34/35 Golgi Transport Complex Is Related to the Exocyst, Defining a Family of Complexes Involved in Multiple Steps of Membrane Traffic  James R.C. Whyte, Sean Munro  Developmental Cell  Volume 1, Issue 4, Pages 527-537 (October 2001) DOI: 10.1016/S1534-5807(01)00063-6

Figure 1 The Yeast Gene DOR1 Encodes a Protein Related to Sec5, a Component of the Exocyst BLAST searches of the GenBank database with the protein sequence encoded by the yeast gene DOR1 (YML071c) identified a single close relative from human (Hs), C. elegans (Ce), D. melanogaster (Dm), and A. thaliana (At). An iterative PSI-BLAST search with human Dor1p revealed homology to a human protein of unknown function and its relatives in other species [ANG2 (c11orf2), E = 6 × 10−9 after one iteration], and then to rat Sec5 (E = 7 × 10−4 after three iterations). The homology is greatest in the N-terminal half of the proteins, and these regions are shown aligned using CLUSTAL W (Thompson et al., 1994), residues shaded if identical (black) or conserved (gray) in at least three proteins. For the Dor1 and ANG2 families the species ORF names for each gene are stated. The sequence of human Dor1 was assembled from overlapping ESTs identified by homology to murine Dor1 (GenBank accession no. BAA95093) Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)

Figure 2 Dor1p Is Associated with Seven Other Proteins Eluates from IgG-Sepharose beads incubated with lysates from 40 g of yeast expressing a protein A tagged form of Dor1p or from the same mass of a control strain (–). Eluted proteins were separated by gel electrophoresis and stained with Coomassie blue. The indicated bands were identified by mass spectrometry of tryptic fragments. Sec34p was mainly present as two bands smaller than its predicted molecular weight, but this is apparently due to postlysis proteolysis as a full-length version of the protein, which comigrates with Cod1p and Cod2p, was found in smaller scale precipitations performed in the absence of detergent. Two of the minor bands corresponded to Cod1p and Cod2p (*) and presumably represent proteolysis during isolation. Ssa1/2p are hsp70s, and frequent contaminants of precipitations, with a comigrating band also being present in the control lane Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)

Figure 3 Protein A-Tagged Cod1–5p Are Associated with Dor1p and Sec35p (A) Protein blots of total cellular protein, or of IgG-Sepharose precipitates, from yeast strains in which the only copy of the indicated gene is tagged with protein A, or no gene is tagged (control). The strains also contain a plasmid expressing HA-tagged Dor1p. (B) Protein blots of the IgG-Sepharose precipitates from the same strains as in (A), but probed with rabbit anti-Sec35p, which also binds to the protein A fusions (*). (C) Electron micrograph of negatively stained Dor1p-containing particles. IgG-Sepharose precipitates were prepared as in A, but the protein eluted in native conditions with TEV protease, applied to grids and stained with 2% uranyl acetate for microscopy Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)

Figure 4 Deletion of COD3 Results in Aberrant Accumulation of Internal Membranes Electron micrographs of sections of the indicated yeast strains following permanganate fixation and embedding (Kaiser and Schekman, 1990). The Δcod3 strain contains many small darkly staining structures and has fragmented vacuoles. This resembles sec35-1 cells fixed an hour after being elevated to the nonpermissive temperature. In contrast, Δdor1 and Δcod2 appear similar to wild-type (BY4741), and a similar result was obtained for Δcod4 and Δcod5 (data not shown). Scale bar, 2 μm Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)

Figure 5 Membrane Trafficking Phenotypes of Strains Lacking DOR1 and COD1–5 (A) Anti-myc protein blots of total cellular protein from yeast strains expressing myc-tagged version of the secreted glycoprotein invertase. Δmnn9 mutants lack all of the mannan structure that is attached in the Golgi to the N-glycans of invertase and other glycoproteins. (B) Confocal micrographs of live cells of the indicated yeast strains expressing a GFP-tagged version of the v-SNARE Snc1p. The fusion is expressed from a CEN plasmid that gives no greater than twice the level of the endogenous protein (Lewis et al., 2000). Scale bar, 2 μm Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)

Figure 6 Human Homologs of Dor1p and Cod1p Are Found on the Golgi Confocal micrographs of COS cells transfected with plasmids encoding triple myc-tagged cDNAs of human Dor1 or Cod1. Cells were fixed, permeabilized, and stained with antibodies against the myc tag and the indicated Golgi marker. At higher expression levels, the tagged proteins accumulated throughout the cytoplasm. Scale bars, 5 μm Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)

Figure 7 Homology between Components of the Sec34/35 Complex and Other Transport Complexes (A) Alignment of the N-terminal amphipathic helical regions of the indicated proteins: Dor1, Cod4, and Sec35p from the Sec34/35 complex, Sec5, Sec8, and Exo84 from the exocyst, Vps53 and Vps54, the putative GTC components ldlCp and ldlBp, and a protein of unknown function, ANG2. In each case, the human homolog is shown, with the exception of yeast Sec35p, which does not have clear homologs in higher eukaryotes. Homology was identified by PSI-BLAST searches with full length human Dor1, Sec5, or ldlCp. In each case, significant homology (E < 0.005) was found to the other two families, plus those of GTC-90 (Cod4), ldlBp, and ANG2 (using 5, 7, and 5 iterations for Dor1, Sec5, and ldlCp respectively). Vps53 and Vps54 were found in addition when the search was with the first 200 residues of ldlCp. The homologous regions were aligned in CLUSTAL W, and residues shaded if identical (black) or conserved (gray) in at least three proteins. Gray bars show the regions predicted to form coiled-coil (the hydrophobic heptad repeat indicated by black circles), with the region between predicted by JPred (Cuff et al., 1998) to be nonhelical, or even extended, suggesting it could form a loop. (B) Alignment of the N-terminal portions of Sec34, Cod2, and Exo70 family members from the species indicated as in Figure 1. A PSI-BLAST search with C. elegans Exo70 (C43E11.8) showed highly significant homology to Sec34 and Cod2 family members after three iterations. The gene names for the Sec34 and Cod2 families are in Table 1. For Exo70, Dm is CG7127 and At is F12E4.320. Alignment and shading as in (A). (C) Alignment of portions of Sec3 and Vps52 family members from the indicated species. A PSI-BLAST search with full length Drosophila Sec3 finds a member of the Vps52 family after a single iteration (E < 0.001), and the rest after a further iteration (E = 2 × 10−27 for human Vps52). Species indicated as Figure 1, genes for Sec3 being Ce, F12E4.7 and At, F16N3.18; and for Vps52, Dm, CG7371; Ce, F08C6.3; and At, F3I17.5 Developmental Cell 2001 1, 527-537DOI: (10.1016/S1534-5807(01)00063-6)