Proteasearrest protease inhibitor cocktail

DOPC and DOPS were purchased from Avanti Polar Lipids (Alabaster, AL, USA). ATP and ascorbic acid were purchased from Sigma-Aldrich, CHAPS from Anatrace (Maumee, OH, USA), ProteaseARREST protease inhibitor cocktail from G-Biosciences (St. Louis, MO, USA) and 1D4 peptide from Biomatik (Kitchener, ON, USA). MG132 was from ApexBio (Houston, TX, USA). HEK293T and HeLa cells were obtained from American Type Culture Collection through Cedarlane Laboratories (Burlington, Ontario, Canada). The Rho1D4 antibody initially produced in house (Hodges et al., 1988 (link); Molday and Molday, 2014 (link)) was obtained from the University of British Columbia (https://uilo.ubc.ca/industry-partners/access-ubc-technologies). The primary antibody against tubulin was from Abcam (ab15568; Toronto, Ontario, Canada) and the antibody against LAMP2 was from Santa Cruz Biotechnology (sc-5571; Dallas, TX, USA). Primary antibodies against ATP8A2 (ATP6C11 and Cdc50-7F4) have been described previously (Coleman et al., 2009 (link); Coleman and Molday, 2011 (link)). Restriction enzymes, T4 DNA ligase, Antarctic Phosphatase and Phusion polymerase were acquired from New England Biolabs (Whitby, Ontario, Canada). Primers used for mutagenesis were ordered from Integrated DNA Technologies (Coralville, IA, USA).

Wild type Arabidopsis thaliana ecotype Columbia was grown in the University of Guelph phytotron under a regime of 16 h light/8 h dark, 22°C/18°C, ambient humidity, 150 μmol photons m⁻² s⁻¹ light intensity in “L4 Sunshine Mix” soil for 22 days. Chloroplast extracts were prepared by harvesting mature rosette leaf tissue 1 h before light (end of night), and submerging immediately in pre-cooled grinding buffer (50 mM HEPES-KOH, pH 7.5; 330 mM sorbitol; 1 mM MgCl₂; 1 mM MnCl₂), on ice. Leaves were homogenized and filtered through Miracloth (CalBiochem Cat No. 475855), before centrifuging at 1,000 g at 4°C for 8 min. The chloroplast-enriched pellet was resuspended and lysed in rupturing buffer (RB) (20 mM Tricine, pH 7.5; 7.5 mM MgCl₂; 1 mM DTT and 1x ProteaseArrest protease inhibitor cocktail [G-Biosciences Cat No. 786-332)]. To remove starch and membranes, lysed chloroplasts were centrifuged at 14,000 g for 10 min at 4°C, and the supernatant flash-frozen in liquid nitrogen and stored at −80°C until needed. Maize endosperm amyloplasts were prepared from fresh kernels ~22 days after pollination using a modified method previously described by Tetlow et al. (2008 (link)). The plastids were osmotically lysed with rupturing buffer, described above.