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

The exocyst is required for endocytosis.

<p>(A) Uninduced and induced Sec15 and Exo99::RNAi cells were allowed to accumulate FITC-conjugated ConA at the indicated temperatures. Fixed cells counterstained with DAPI (blue) and anti-p67 antibody for the lysosome (green). Both Sec15 and Exo99::RNAi lines show a defect in delivery of ConA to the lysosome: at 12°C and 37°C ConA remains predominantly localised at the flagellar pocket. (B) Dynamics of ConA uptake at 37°C 48h post RNAi induction. In control cells, ConA accumulates in a p67-positive compartment within 30 min. By contrast Sec15 and Exo99::RNAi cells exhibit a marked delay in delivery of ConA to the lysosome. In all images DAPI was used to visualise DNA (blue). Scale bar, 5 μm. (C) Quantitation of ConA lysosomal delivery at 37°C 48h post induction. N = 25 cells per time point per duplicate experiment. (D) Representative electron micrograph of curved flagellar pocket membrane coated by clathrin. Arrowheads point at clathrin coat.</p

The exocyst functions in endocytosis in human cells.

<p>(A) Immunoblots of lysates prepared from HeLa cells treated with either scrambled or EXOC6 siRNA smart pools as shown in panel B, probed with anti-EXOC6 or anti-GAPDH as indicated. Knockdown levels of EXOC6 were quantified from 3 experiments of this type and are presented as a ratio of EXOC6/GAPDH signals. (B) HeLa cells depicting transferrin uptake. Cells treated with either scrambled or EXOC6 siRNA were imaged after the uptake of labelled transferrin (10 min). Representative fields of cells from 3 independent replicates. (C) The fluorescence intensity of >100 cells for each condition was determined using ImageJ and the data compared using an unpaired t test. *** P value < 0.0001. (D) The image intensity of >100 cells of each condition were binned into two groups, those with a signal intensity >500 arbitrary units, and those <500. Knockdown of EXOC6 was found to significantly increase the fraction of cells with a signal intensity <500 (** P value <0.01), consistent with a decrease in transferrin endocytosis.</p

The exocyst is not required for endomembrane system maintenance or morphology.

<p>Immunofluorescence microscopy analysis of selected organelle markers or trafficking pathways for Sec15::RNAi and Exo99::RNAi cells at 24 (D1) and 48h (D2) post induction. Cells were stained with specific antibodies against GRASP (red), p67 (green), clathrin heavy chain (red) or Rab11 (red) and counter-stained with DAPI to visualise DNA (blue). The morphology and protein expression levels of all molecular markers remained stable. Scale bar, 5 μm.</p

Identification of nine exocyst subunits in trypanosomes.

<p>(A) Immunoisolation from PCF cells constitutively expressing Sec15::GFP or Exo99::GFP. Pullouts were performed with the following buffers; (1) 20mM HEPES pH 7.4, 500mM NaCl 5% Triton and protease inhibitors; (2) 20mM HEPES pH 7.4, 250mM NaCitrate 5% CHAPS and protease inhibitors. (B) Immunofluorescence microscopy of PCF cells expressing Sec15::GFP and Exo99::GFP (green), BSF cells expressing Sec15::HA and Exo99::HA (red) and PCF cells co-expressing Sec15::GFP and Exo99::HA. DAPI was used to visualise DNA (blue). Scale bar, 5 μm. In both lifecycle stages Sec15 and Exo99 localise to the region between the nucleus and kinetoplast where the organelles of the endocytic and secretory pathways are found. (C) Immunofluorescence of PCF cells expressing Sec5::GFP and Exo84::GFP (green). Sec5 and Exo99 localise to the same region which indicates that they are part of the same complex. (D) Predicted secondary structure of Exo99 according to PSIPRED. The horizontal black line represents the polypeptide span with the N-terminus to the left, the y-axis indicates the confidence score of predicted secondary structure. Predicted α-helix are shown in red, predicted β-sheet in blue.</p

The exocyst is required for normal membrane trafficking.

<p>Representative transmission electron micrographs showing the effect of exocist subunit ablation 48h post-RNAi induction in BSF cells. The steady-state flagellar pocket has a small overall volume (A); ablation of Exo99 and Sec15 causes pocket enlargement (B, E), over-production of large VSG-coated vesicles inside the flagellar pocket (C, F, G), failure in cytokinesis (as illustrated by multiple nuclei in panels D and I) and ER hypertrophy (H). Despite large flagellar pocket volumes, indicative of endocytosis defect, clathrin is recruited to the surface membrane and able to assemble into coated pits and lattices (B, F).</p