35s methionine

The ³⁵S-methionine incorporation was based on the protocol described in ref. 54 (link). Briefly, Jazz-Mix Drosophila medium (Fisher Scientific, cat. no. AS153) was supplemented with 100 μCi ³⁵S-methionine per ml of food (American Radiolabeled Chemicals, St Louis, USA; 1 mCi/37 MBq, ARS 0104A). Ten flies were transferred to each vial containing radioactive food. After 24 h of feeding, flies were transferred to nonradioactive food for 30 min in order to purge undigested ³⁵S-methionine radioactive food out of the intestines. Flies were then homogenized in 200 μM 1% SDS and heated for 5 min at 95 °C. Samples were centrifuged for 5 min at 13,000g at 4 °C and supernatant was retained. Proteins were precipitated by the addition of the same volume of ice-cold acetone and incubation on −20 °C for 1 h. Samples were then centrifuged at 13,000g for 15 min at 4 °C, supernatant was removed and the pellet was resuspended in 200 μl 4 M guanidine-HCl. An amount of 100 μl of each sample was used for scintillation counting, and the remaining 100 μl was used for Bradford assay to determine total protein concentrations. Counts per minute (CPM) measurements were normalized to total protein for each sample.

Cells harbouring an appropriate combination of plasmids encoding an RseP derivative and a model substrate were grown at 30°C in M9 medium supplemented with 20 μg/ml of each of 18 amino acids (except Met and Cys), 2 μg/ml thiamine and 0.4% glucose to a mid-log phase and were induced with 1 mM IPTG and 5 mM cAMP for 10 min. The cells were then labelled with 370 kBq/ml [³⁵S]-methionine (American Radiolabeled Chemicals, St. Louis, MO) for 30 s. Chasing was performed using 200 μg/ml unlabelled methionine for the indicated periods. Proteins were precipitated using 5% (final concentration) TCA, washed with acetone, dissolved in 50 mM Tris–HCl (pH 8.1) containing 1 mM EDTA and 1% SDS and immunoprecipitated with anti-HA antibody, as described previously (Akiyama et al., 2004 (link)). Labelled proteins were separated by SDS-PAGE and were visualised and quantified using a phosphor imager (BAS1800) (Fujifilm).

Fao cells growing in DMEM supplemented with 10% FBS in 6-well plate were washed twice with PBS, incubated in cysteine- and methionine-free DMEM (Gibco) supplemented with 10% FBS that had been dialyzed for 1 hr in excess PBS with a Slide-A-Lyzer dialysis cassette (Thermo Fisher Scientific). Cells were then pulse-labeled for 1 hr by adding 100 μCi/ml ³⁵S-methionine plus ³⁵S-cysteine (American Radiolabeled Chemicals). To chase the ³⁵S-labeled proteins, cells were washed twice and further incubated for 1 hr with DMEM supplemented with 10% FBS and 10 mM methionine. ³⁵S-labeled cells were harvested and incubated for 5 min in buffer H containing 50 μg/ml digitonin at room temperature as described (Natsuyama et al., 2013 (link)). After centrifugation at 20,000 g for 30 min at 4°C, cytosolic and organelle fractions were subjected to immunoprecipitation with antibodies to catalase and DHAPAT as described (Tsukamoto et al., 1990 (link)). ³⁵S-labeled proteins were separated by SDS-PAGE and detected with an Autoimaging analyzer (Typhoon FLA-9500; GE Healthcare).