L 35s methionine

The concentrations of bioavailable phosphate, methionine, ATP and microbial uptake rates were estimated at 20, 13 and 17 stations, respectively, on the 2010 cruise (Supplementary Figs 4–5) using an isotope dilution, concentration series bioassay2 (link)7 (link). L-[35S] methionine (specific activity >1,000 Ci mmol 1⁻¹, Hartmann Analytic GmbH, Braunschweig, Germany) was added at a concentration of 0.05 nM and diluted with unlabelled L-methionine (Sigma Aldrich, Dorset, UK) using a dilution series spanning the range 0.05–1.0 nM. [α ³³P]-ATP (specific activity >3,000 Ci mmol 1⁻¹, Hartmann Analytic) was added at a concentration of 0.05 or 0.1 nM and diluted with non-labelled ATP–disodium salt hydrate (Sigma Aldrich) using a dilution series in the range 0.1–2.0 nM. ³³P-phosphate tracer (specific activity 100 TBq mM⁻¹, Hartmann Analytic) was added to samples at <0.05 nmol l⁻¹ final concentration and diluted with unlabelled orthophosphoric acid using a dilution series spanning the range of 0.4–4.0 nM. On the 2012 cruise only the bioavailable concentration and microbial uptake of phosphate were estimated at 11 stations (Supplementary Fig. 2). Multiple replicated 1.6-ml samples were incubated in crystal clear screw cap microtubes (Starlab, Milton Keynes, UK) at in situ temperature in the dark and fixed with 1% (w/v) PFA.

To analyze the subcellular localization of PLAC1, an in silico-predicted signal peptide, with a cutting site between amino acid (aa) 22 to 23, was designed to produce a truncated form of PLAC1 in vivo. In vitro–coupled transcription/translation of proteins was performed using the TNT Quick Coupled Transcription/Translation Reticulocyte Lysate System according to the manufacturer´s protocol (Promega, Madison, WI, USA), using radioactive labeled L-[35S] methionine (Hartmann Analytic GmbH, Braunschweig, Germany). Reactions were incubated for 90 minutes at 30°C. Equal amounts of sample were then treated with 4× SDS-lysis buffer and analyzed by SDS-PAGE and autoradiography.