Mak030

Cardiac myofibrils were prepared, endogenous RLCs exchanged with either wildtype RLC or RLCs crosslinked to bifunctional rhodamine, and the ATPase activity under relaxing conditions determined as previously described^{22 (link)}. Briefly, cardiac myofibrils (CMFs) were prepared by homogenising freshly frozen ventricular tissue samples in myofibril buffer (composition in mmol L⁻¹: 20 imidazole, 75 KCl, 2 MgCl₂, 2 EDTA, 1 DTT, 1% (v/v) Triton X-100, pH 7.4, protease inhibitor cocktail (ROCHE), PhosStop cocktail (ROCHE)) followed by centrifugation at 5000 g for 5 min at 4 °C. CMFs were washed and homogenised three more times in the same buffer without Triton X-100. Endogenous RLCs were extracted from CMFs and reconstituted with recombinant proteins using the same protocol as described above for trabeculae. CMFs were washed three times in ATPase assay buffer (composition in mmol L⁻¹: 20 MOPS, 35 NaCl, 5 MgCl₂, 1 EGTA, 1 DTT, pH 7.0). Reactions were started by the addition of 2.5 mmol L⁻¹ ATP and samples were quenched with 0.5 volumes ice cold 25% (w/v) TCA solution. Samples were kept on ice at all times, diluted with double-deionized water and inorganic phosphate content measured using the malachite green assay according to manufacturer’s instructions (SIGMA, GILLINGHAM SP8 4XT, United Kingdom; MAK030).

xlCHPT1 activity was measured using an absorbance-based coupled-enzyme assay (Supplementary Fig. 2b). All reaction assays were done in a buffer with 20 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% GDN, 5 mM MgCl₂. In single point activity assay, final concentrations of CDP-choline and 1,2-sn-diacylglycerol were 0.2 mM and 0.25 mM, respectively. Reactions were initiated with the addition of 10 μg purified xlCHPT1 protein. After reaction at 37 °C for 15 mins, reactions were stopped by heating up at 95 °C for 5 min. After removing the precipitated protein with centrifugation, purified yeast pyrimidine 5′-nucleosidase SDT1 was added into the supernatant, then react at 37 °C for 20 mins. Finally, phosphate dye (Sigma, MAK030) was added to the reaction solution and A_650 nm was measured in 96 plates. When CDP-choline or CDP-ethanolamine concentrations are varied, DAG concentration is fixed at 250 μM. The initial rate versus different concentrations of CDP-choline and CDP-ethanolamine can be fit with a Michaelis–Menten equation.

A commercially available phosphate colorimetric kit (Sigma-Aldrich; MAK030) and a previously published protocol (67 (link)) were utilized to measure intracellular inorganic phosphate (P_i) levels in wild-type and DAP-B1 cultures. Overnight cultures were diluted to an OD₆₀₀ of 0.01 in 50 ml of prewarmed BHI, followed by incubation at 37°C with shaking at 100 rpm. The phosphate levels were measured at four time points: 1, OD₆₀₀ = 0.4 to 0.5; 2, OD₆₀₀ = 0.5 to 0.6; 3, OD₆₀₀ = 0.6 to 0.7; and 4, OD₆₀₀ = 0.7 to 0.8. For each time point, 100-μl culture was serially diluted in 0.9% sterile NaCl and spotted on BHI plates for CFU determination. Also, 1 ml of the culture was incubated on ice for 5 min and then pelleted at 13,300 × g for 2 min at 4°C. The pellet was washed twice in 1 ml of double-distilled water, resuspended in 0.5 ml of double-distilled water, and then disrupted three times (Fast-Prep-24; MPBio) at 6.5 m/s for 30 s. The homogenized samples were centrifuged at 13,300 × g for 15 min at 4°C. Twenty-five μl of the supernatant was diluted with 25 μl double-distilled water, and the P_i levels were measured per the manufacturer's instructions. The phosphate levels were normalized by determining the CFU. A one-tailed Student t test was used to assess significance from three independent trials.

The concentration of poly-P was measured using a spectrophotometer after staining poly-P in toluidine blue [13 (link)]. The microalgal culture (10 ml) was centrifuged at 2,350 ×g for 5 min to remove the supernatant, and the cell pellet was washed twice with 500 μl sterilized water. After measuring the weight of the cell, 600 μl sterilized water was added to resuspend the cells. After ultrasonication for 5 min, the mixture was placed in a water bath at 100°C and boiled for 2 h. Thereafter, it was cooled at 25°C, and 600 μl of a 24:1 (v/v) chloroform:isoamyl alcohol mixture was added. After centrifugation at room temperature for 15 min, the supernatant was transferred to a new tube. Toluidine blue solution (3 ml) and acetic acid solution (0.2 N) were then added to the tubes. The absorbance of the samples was measured at 630 nm using a microplate reader (Victor X3, PerkinElmer, Inc., USA).
The amount of inorganic phosphate in the medium absorbed by the cells was measured using a phosphate colorimetric analysis kit (MAK030; Sigma-Aldrich). Absorbance was measured in the same way as described above.