Overlapping binding of TAX1BP1 zinc finger domains with ubiquitin and myosin VI.

<p>(<b>A</b>) Domain organisation of TAX1BP1 and designated point mutations. LIR, LC3-interaction region; ZF, zinc-finger; SKICH, SKIP carboxyl homology. (<b>B</b>) Ribbon presentation of the structures of the TAX1BP1 zinc fingers together with two molecules of ubiquitin. The residues are colored according to the extent of the chemical shift changes observed where blue represents small or no chemical shift changes observed, and red represents the maximum chemical shift changes observed for the backbone amides. The side chains of the residues in TAX1BP1 that when mutated abolish ubiquitin binding are displayed and labelled. (<b>C</b>) RPE cells were transfected with GFP empty vector, with GFP-TAX1BP1 wild-type, GFP-TAX1BP1 ZF1 mutant (Q743A/E747K), GFP-TAX1BP1 ZF2 mutant (Q770A/E774K) or the Q743A/E747K/Q770A/E774K double zinc finger mutant of GFP-TAX1BP1 followed by GFP immunoprecipitation and pull-down with K63-linked polyubiquitin (2–7). Western blot analysis was performed and immunoblotting against the indicated proteins. (<b>D</b>) Mammalian 2-hybrid assay in CHOK.1 cells with myosin VI tail as bait and TAX1BP1 full-length wild-type and mutants as prey. Results are represented as the normalised luciferase activity against a bait only control.</p

TAX1BP1 interacts with LC3B as well as LC3C and requires its zinc finger domains to localise to ubiquitylated Salmonella.

<p>(<b>A</b>) Mammalian 2-hybrid assay in CHOK.1 cells with LC3A, B, or C and GABARAP, GABARAPL1 or L2 as bait and TAX1BP1 full-length wild-type as prey. Results are represented as the normalised luciferase activity against a bait only control. <b>(B)</b> Mammalian 2-hybrid assay in CHOK.1 cells using LC3B or LC3C as bait and full-length TAX1BP1, NDP52, optineurin wild-type and LIR mutants as prey. Results are represented as the normalised luciferase activity against a bait only control. (C) Mammalian 2-hybrid assay in CHOK.1 cells with LC3B as bait and TAX1BP1 full-length wild-type and mutants as prey. Results are represented as the normalised luciferase activity against a bait only control. (<b>D</b>) HeLa cells transfected with GFP-TAX1BP1 wild-type or V144S LIR mutant, Q743A/E747K/Q770A/E774K double zinc finger mutant, and V144S/Q743A/E747K/Q770A/E774K LIR and double zinc finger mutant were infected with mCherry expressing Salmonella for 1 hour prior to saponin extraction and fixation. Cells were immunostained for GFP and ubiquitin and processed for confocal microscopy. Nuclei are labelled with Hoechst (blue). Scale bar, 20 μm.</p

Suppression of myosin VI expression leads to a hyper-proliferation of Salmonella and an accumulation of ubiquitylated Salmonella within LC3-positive autophagosomes.

<p>(<b>A</b>) HeLa cells following myosin VI siRNA or TAX1BP1, NDP52, OPTN (TNO) siRNA transfection were subjected to Salmonella infection for the indicated time points. Gentamicin protection assays were performed and colonies were counted at each time point. Results are represented as the fold replication from 2 hours, which represents the amount of bacterial proliferation. Results are from >3 independent experiments and error bars represent s.d. Western blot analysis was performed on whole cell lysates and immunoblotting was performed to the indicated proteins. (<b>B</b>) HeLa cells mock, myosin VI siRNA, or TNO siRNA treated were infected with mCherry expressing Salmonella for 8 hours, followed by processing for confocal microscopy. Cells were immunostained for ubiquitin (green). Nuclei were labelled with Hoechst (blue). The % of infected cells with ubiquitin (+) Salmonella was quantified. The results depict >3 independent experiments and the error bars represent the s.d. (<b>C</b>) HeLa cells transfected with myosin VI or TNO siRNA were infected with BFP-expressing Salmonella for 8 hours. Cells were processed for confocal microscopy and immunostained for LC3 (green) and ubiquitin (red). Scale bar, 20 μm. Quantitation at 4 and 8 hours post-infection, with BFP-expressing Salmonella, of the % of ubiquitin (+) Salmonella which are LC3 (+). Results represent 3 independent experiments and the error bars indicate the s.d.</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 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

Quantitative proteomics by SILAC identifies altered surface protein expression mediated by exocyst knockdown.

<p>(A) Volcano plot of protein abundance changes at 36h post Sec15 RNAi induction. -log₁₀ transformed SILAC ratios are plotted against -log₁₀ transformed standard deviation. Data points representing protein groups significantly altered after 36 hours are labeled; ISG65 and ISG75 paralogs in dark green, other potential surface proteins in light green, mitochondrial proteins in red, cytoplasmic proteins in blue, lysosomal proteins in yellow, others in grey. AQP1: Aquaporin1, TryS: trypanothione synthetase; PTP1 interactor: Protein-tyrosine-phosphatase1-interacting protein; PAD1: Protein associated with differentiation 1; CoA transferase: Succinyl-coA:3-ketoacid-coenzyme A transferase; GCVL-2: Dihydrolipoyl dehydrogenase. (B) Predicted cellular localisation of proteins upregulated in Sec15 RNAi cells 36h post induction as reported by GO terms or as predicted by TMHMM2 for <i>trans-</i>membrane domains and PredGPI for GPI-anchor addition C-terminal signal sequences [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006063#ppat.1006063.ref089" target="_blank">89</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006063#ppat.1006063.ref090" target="_blank">90</a>]. Numbers of proteins in each category are in parenthesis.</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