Similarity to exocyst subunits of known structures.

<p>HMM P-values of the comparisons are shown in bold, with the range of the aligned residues in parentheses below. Yeast protein lengths are indicated by the number of amino acids (aa). SGD identifiers are indicated in the first column of the table, and PDB identifiers are indicated in the second row of the table. Blank cells have P-values>1.</p

Sec10(145–827) is functional for protein-protein interactions <i>in vitro</i>.

<p>Sec10(145–827) binds to MBP-Sec6p, MBP-Exo70p (residues 63–623) and MBP-Exo84p (residues 523–753), but not to MBP alone. The MBP, MBP-tagged Sec6p, Exo70p and Exo84p proteins were immobilized on amylose resin and incubated with Sec10(145–827). Equivalent volumes of the bound fractions [10% of the input of Sec10(145–827) is shown in the first lane as a control for the amount of Sec10(145–827) bound] were analyzed on denaturing SDS-PAGE gels. His₆-tagged Sec10(145–827) and MBP-tagged partners were detected by Western blot analyses using α-His₅ and α-MBP antibodies, respectively.</p

Similarity between full-length exocyst subunits.

<p>HMM P-values of the comparisons are indicated for the full length proteins. SGD identifiers are indicated in the first column of the table.</p

Recombinant Sec10(145–827) is soluble.

<p>Several Sec10p truncation constructs designed using secondary structure predictions are not generally soluble. (<i>A</i>) Secondary structure prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Jones1" target="_blank">[41]</a> and schematic of several representative N- and C-terminal truncations tested. The secondary structure prediction is schematically depicted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone-0004443-g001" target="_blank">Figure 1</a>. Truncations 1–589 and 590–871 were derived from dominant negative constructs described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Roth1" target="_blank">[27]</a>. (<i>B</i>) <i>E. coli</i> cells were transformed with Sec10p truncation variants cloned with an N-terminal His₆-tag in the vector pET15b (Novagen). Expression was induced by addition of IPTG to 0.1 mM, and growth was continued at 15°C for 14–18 h. Cells were pelleted, lysed and the insoluble (P) material was separated from the soluble material (S) by centrifugation; these were run on a 10% SDS-PAGE gel and stained with Coomassie blue dye. Asterisks indicate the migration of each construct. For each construct except Sec10(145–827), very little of the His₆-tagged protein was in the soluble fraction. Although the Sec10(75–859) construct initially appeared promising, it was sticky and aggregated after partial purification on Ni-NTA resin. The right hand lane contains Sec10(145–827) after purification by Ni-NTA resin and gel filtration chromatography.</p

The exocyst subunits have similar helical bundle structures.

<p>(<i>A</i>) The known structures of the exocyst subunits are shown: Exo70p (PDB ID 2B1E), Exo84CT (PDB ID 2D2S), Sec15CT (PDB ID 2A2F), Sec6CT (PDB ID 2FJI). Molecular graphics were generated with PyMOL (<a href="http://pymol.sourceforge.net/" target="_blank">http://pymol.sourceforge.net/</a>). Exo84CT is aligned with the N-terminal helical bundles of Exo70p, while Sec15CT and Sec6CT are aligned with the C-terminal bundles of Exo70p. (<i>B</i>) Secondary structure predictions for all of the exocyst subunits. The black horizontal lines represent the sequence of each yeast exocyst subunit. The predicted α-helices (magenta) and β-strands (cyan) are indicated by vertical bars above each line. The height of the bars is proportional to the confidence of the secondary structure prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone.0004443-Jones1" target="_blank">[41]</a>. Red blocks underline regions of the known structures. Green blocks underline the best hits to exocyst structures (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004443#pone-0004443-t001" target="_blank">Table 1</a>).</p

Predicted Secondary Structure Maps of the Nup84 Subcomplex Proteins

<p>Thin horizontal lines represent the primary sequence of each protein; secondary structure predictions are shown as columns above each line for β-strands (β-propellers; cyan) and α-helices (α-solenoids; magenta). The height of the columns is proportional to the confidence of the secondary structure prediction (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-McGuffin2" target="_blank">McGuffin et al. 2000</a>). The modeled regions are indicated above each sequence by horizontal dark bars, corresponding to the models in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-g001" target="_blank">Figure 1</a>. Proteolytic cleavage sites are identified by small, medium, and large arrows for weak, medium, and strong susceptibility sites, respectively. Where necessary, uncertainties in the precise cleavage positions are indicated above the arrows by horizontal bars.</p

Ribbon Representation of Nup Models

<p>β-sheets (β-propellers) are colored cyan and α-helices (α-solenoids) are colored magenta. Gray dashed lines indicate regions that were not modeled. Arrowheads indicate the positions of high proteolytic susceptibility (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-g002" target="_blank">Figures 2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-g003" target="_blank">3</a>).</p

The Nup84 Complex and Coated Vesicles Share a Common Architecture

<p>A diagram showing the organization of the clathrin/AP-2 coated vesicle complex is shown at left; the positions of clathrin and the adaptin AP-2 large subunits (α, β2 plus “ear” domains) and small subunits (σ, μ) are indicated. β-propeller regions are colored cyan, α-solenoid regions are colored magenta, and sample ribbon models for each fold are shown in the center. The variants of each fold that are found as domains in major components of the three kinds of vesicle-coating complexes and the yNup84 subcomplex are listed on the right. The -N and -C indicate amino-terminal and carboxyl-terminal domains, respectively. The classification of these domains is based on X-ray crystallography data (clathrin, α-adaptin, β2-adaptin [PDB codes 1gw5, 1bpo, 1b89 (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-ter1" target="_blank">ter Haar et al. 1998</a>; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-Collins1" target="_blank">Collins et al. 2002</a>)]), by the detailed homology modeling presented here (yNup84 complex proteins; ySec13 also in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-Saxena1" target="_blank">Saxena et al. [1996]</a>), or by sequence homology or unpublished secondary structure prediction and preliminary analyses (COPI I (sec31) complex proteins [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-Schledzewski1" target="_blank">Schledzewski et al. 1999</a>], Sec31).</p

Proposed Model for the Evolution of Coated Vesicles and Nuclear Pore Complexes

<p>Early eukaryotes (left) acquired a membrane-curving protein module (purple) that allowed them to mold their plasma membrane into internal compartments and structures. Modern eukaryotes have diversified this membrane-curving module into many specialized functions (right), such as endocytosis (orange), ER and Golgi transport (green and brown), and NPC formation (blue). This module (pink) has been retained in both NPCs (right bottom) and coated vesicles (left bottom), as it is needed to stabilize curved membranes in both cases.</p

Proteolytic Domain Map of the Yeast Nup84 Subcomplex Proteins

<p>Immunoblots of limited proteolysis digests for Protein A-tagged versions of each of the seven nups in the yNup84 subcomplex. Each protein is detected via its carboxyl-terminal tag; thus, all the fragments visualized are amino-terminal truncations (except for the full length proteins, which are indicated by arrowheads). The fragments of the Asp-N and Lys-C protease digests depicted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-g002" target="_blank">Figure 2</a> are labeled with letters (A, B, C…) that correspond to those in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020380#pbio-0020380-t002" target="_blank">Table 2</a>, and the terminal Protein A fragments are labeled with an X (the Protein A tag is resistant to proteolysis). The sizes of marker proteins are indicated in kilodaltons (kDa) to the right of the gel.</p