Conservation of Helical Bundle Structure between the Exocyst Subunits

Background: The exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies. Methodology/Principal Findings: Using advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60 % a-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners. Conclusions/Significance: Although all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folde

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

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

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

Functional homology of mammalian syntaxin 16 and yeast Tlg2p reveals a conserved regulatory mechanism

Membrane fusion in all eukaryotic cells is regulated by the formation of specific SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. The molecular mechanisms that control this process are conserved through evolution and require several protein families, including Sec1p/Munc18 (SM) proteins. Here, we demonstrate that the mammalian SNARE protein syntaxin 16 (Sx16, also known as Syn16) is a functional homologue of the yeast SNARE Tlg2p, in that its expression fully complements the mutant phenotypes of tlg2Δ mutant yeast. We have used this functional homology to demonstrate that, as observed for Tlg2p, the function of Sx16 is regulated by the SM protein Vps45p. Furthermore, in vitro SNARE-complex assembly studies demonstrate that the N-terminal domain of Tlg2p is inhibitory to the formation of SNARE complexes, and that this inhibition can be lifted by the addition of purified Vps45p. By combining these cell-biological and biochemical analyses, we propose an evolutionarily conserved regulatory mechanism for Vps45p function. Our data support a model in which the SM protein is required to facilitate a switch of Tlg2p and Sx16 from a closed to an open conformation, thus allowing SNARE-complex assembly and membrane fusion to proceed