Project Protein Table
Project Peptide Table
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Exocyst Dynamics During Vesicle Tethering and Fusion
The exocyst is a conserved octameric complex that tethers exocytic vesicles to the plasma membrane prior to fusion. Exocyst assembly and delivery mechanisms remain unclear, especially in mammalian cells. Here we tagged multiple endogenous exocyst subunits with sfGFP or Halo using Cas9 gene-editing, to create single and double knock-in lines of mammary epithelial cells, and interrogated exocyst dynamics by high-speed imaging and correlation spectroscopy. We discovered that mammalian exocyst is comprised of tetrameric subcomplexes that can associate independently with vesicles and plasma membrane and are in dynamic equilibrium with octamer and monomers. Membrane arrival times are similar for subunits and vesicles, but with a small delay (~80msec) between subcomplexes. Departure of SEC3 occurs prior to fusion, whereas other subunits depart just after fusion. About 9 exocyst complexes are associated per vesicle. These data reveal the mammalian exocyst as a remarkably dynamic two-part complex and provide important insights into assembly/disassembly mechanisms.
Sample Processing Protocol
Exocyst protein immunoprecipitations were run ~1.5cm into a NuPAGE Bis-Tris gel to remove SDS from the samples, were stained with Novex Colloidal Blue Coomassie stain (ThermoFisher Scientific), and destained in water. Coomassie stained gel regions were cut from the gel and diced into 1mm3 cubes. Proteins were treated for 30 minutes with 45mM DTT, and available Cys residues were carbamidomethylated with 100mM iodoacetamide for 45 min. Gel pieces were further destained with 50% MeCN in 25mM ammonium bicarbonate, and proteins were digested with trypsin (10ng/uL) in 25mM ammonium bicarbonate overnight at 37°C. Peptides were extracted by gel dehydration with 60% MeCN, 0.1% TFA, the extracts were dried by speed vac centrifugation, and reconstituted in 0.1% formic acid.
Data Processing Protocol
Peptides were analyzed by LC-coupled tandem mass spectrometry (LC-MS/MS). An analytical column was packed with 20cm of C18 reverse phase material (Jupiter, 3 μm beads, 300Å, Phenomenox) directly into a laser-pulled emitter tip. Peptides were loaded on the capillary reverse phase analytical column (360μm O.D. x 100μm I.D.) using a Dionex Ultimate 3000 nanoLC and autosampler. The mobile phase solvents consisted of 0.1% formic acid, 99.9% water (solvent A) and 0.1% formic acid, 99.9% acetonitrile (solvent B). Peptides were gradient-eluted at a flow rate of 350 nL/min, using a 110-minute gradient. The gradient consisted of the following: 1-3min, 2% B (sample loading from autosampler); 3-88 min, 2-40% B; 88-98 min, 40-95% B; 98-99 min, 95% B; 99-100 min, 95-2% B; 100-110 min (column re-equilibration), 2% B. A Q Exactive Plus mass spectrometer (Thermo Scientific), equipped with a nanoelectrospray ionization source, was used to mass analyze the eluting peptides using a data-dependent method. The instrument method consisted of MS1 using an MS AGC target value of 3e6, followed by up to 20 MS/MS scans of the most abundant ions detected in the preceding MS scan. A maximum MS/MS ion time of 60 ms was used with a MS2 AGC target of 1e5. Dynamic exclusion was set to 15s, HCD collision energy was set to 28 nce, and peptide match and isotope exclusion were enabled. For identification of peptides, tandem mass spectra were searched with Sequest (ThermoFisher Scientific) against a Mus musculus database created from the UniprotKB protein database (www.uniprot.org). Variable modification of +15.9949 on Met (oxidation) and +57.0214 on Cys (carbamidomethylation) were included for database searching. Search results were assembled using Scaffold 4.3.2. (Proteome Software). For MRM-MS analysis, peptides for each protein were selected based on their appearance in data dependent analyses and then optimized for the most useful transitions to monitor. Heavy-labeled peptide internal standards were synthesized by jpt (SpikeTides TQL peptides, jpt, Berlin, Germany) which contained isotopically-labeled terminal arginine or lysine residues (13C and 15N) and a trypsin-removable C-terminal tag. These isotopically-labeled peptides were digested separately and then spiked into samples at approximately endogenous levels after in-gel digestion of sample proteins. Skyline software (University of Washington, MacCoss lab) was used to set up scheduled, targeted MRM methods monitoring four to five MRM transitions per peptide. A final MRM instrument method including the isotopically labeled standards and encompassing a 9-minute window around the retention time of each peptides was performed using a 40 mm by 0.1 mm (Jupiter 5 micron, 300A) kasil fritted trap followed by a 250 mm by 0.1 mm (Jupiter 3 micron, 300A), self-packed analytical column coupled directly to an TSQ-Vantage (ThermoFisher) via a nanoelectrospray source. Peptides were resolved using an aqueous to organic gradient flowing at 400 nl min-1. Q1 peak width resolution was set to 0.7, collision gas pressure was 1 mTorr, and utilized an EZmethod cycle time of 3 seconds.
Syed Mukhtar Ahmed, Vanderbilt University School of Medicine
Ian G. Macara, Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN, 37240, United States ( lab head )
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