T http://genome.jgi-psf.org/Chlre4/Chlre4.residence.html (protein ID: 522089). The predicted CVDE protein sequences have been confirmed by comparing against every single other and against the cDNA consensus obtained from UCSC/UCLA genome browser at http://genomes.mcdb.ucla.edu. The CDS of the CrCVDEAt gene was then codon-optimized and synthesized for Arabidopsis nuclear/cytoplasmic expression (GenScript). The synthetic CrCVDEAt gene was subcloned into the Gateway vector pDONR221, plus a FLAG-tag was added correct prior to the stop codon by `Round-the-horn’ site-directed mutagenesis (http:// openwetware.org/wiki/ 27Round-the-horn_site-directed_mutagenesis). Sequence encoding the Arabidopsis PSBS transit peptide (initial 54 amino acids) was amplified to replace the predicted native CrCVDE transit peptide (initially 56 amino acids) in versions of each construct using gene SOEing27. The CrCVDEAt gene as well as the FLAG-tagged CrCVDEAt gene have been subcloned into the pEarleyGate100 vector28 and transformed into the Arabidopsis vde1 mutant16 employing the floral dip method29. As a positive control, a vector containing a FLAG-HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptNat Plants. Author manuscript; accessible in PMC 2017 March 12.Li et al.Pagetagged version of the Arabidopsis VDE1 gene30 was also transformed. The transformants have been selected on Murashige and Skoog plates containing 20 g/mL glufosinate ammonium, screened for NPQ capacity with the IMAGING-PAM M-series (Heinz Walz), measured for NPQ induction with an FMS2 fluorometer (Hansatech Instruments) as previously described31, and assayed for the accumulation of zeaxanthin right after high light exposure by HPLC as described26.IL-13 Protein Biological Activity Chlamydomonas cell fractionationHHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptChlamydomonas cells have been grown photoheterotrophically in TAP medium32 to medium logarithmic phase (approximately 5 106 cells mL-1) and harvested by centrifugation at 3,000g for 5 min. Cells have been resuspended in PBS buffer to a density of 2 108 cells mL-1 and broken by FastPrep-24 (MP Biomedicals, Solon, OH) with lysing matrix J at a speed of 4.0 m/sec for 40 sec. Total membrane and total supernatant have been separated by centrifugation at 20,000g, 4 for ten min.IL-13, Mouse Total membranes were washed three instances just before getting resuspended with 1 PBS buffer containing 100 M phenylmethylsulfonyl fluoride (PMSF).PMID:23672196 Samples were then subjected to immunoblot evaluation as described below.Chlamydonas and Arabidopsis thylakoid isolation The Chlamydomonas thylakoid were isolated by a modification from the flotation procedure described previously33. The Chlamydomonas cells have been grown in 400 mL TAP under low light and harvested at mid-logarithmic development phase. The cell pellet was resuspended in 20 mL of 25 mM HEPES (pH 7.5), 0.3 M sucrose, 10 mM CaCl2, ten mM MgCl2 with protease inhibitors. The cells had been broken by passing the resuspended cells via a chilled French pressure cell, and also the homogenate was centrifuged at 18,000 rpm for ten min. The supernatant was discarded along with the pellet was gently resuspended having a paintbrush in 5 mL of 5 mM HEPES (pH 7.5), 1.eight M sucrose, 10 mM CaCl2, ten mM MgCl2. The resuspension was very carefully transferred into a clear tube for SW41 rotor and topped with six mL of five mM HEPES (pH 7.five), 0.5 M sucrose, ten mM CaCl2, 10 mM MgCl2. The tubes had been centrifuged at 38,000 rpm (SW41, four ) for 1 hour. The membrane layer at the interface of two options was meticulously transferred to a 1.5 mL eppendorf tube c.
