Fig. 5. Mechanistic coupling of polyP synthesis and membrane translocation.

a Assay of VTC-catalyzed polyP synthesis. 5 μM protein complex were used and reactions were performed with required times in the presence of 1 mM InsP₆. Wt, Vtc1/Vtc3/Vtc4; Vtc1^TM1,mutant, Vtc1_K24A/R31A/Vtc3/Vtc4; Vtc3^TM1,mutant, Vtc1/Vtc3_{K694A/K698A/R705A/R709A}/Vtc4; Vtc4^TM1,mutant, Vtc1/Vtc3/Vtc4_K622A/R629A; Vtc1^ΔN, Vtc1_Δ1–21/Vtc3 /Vtc4. Source data are provided as a Source Data file. The experiments were repeated three times independently with similar results. b In-cell ³¹P-NMR measurement of vacuolar polyP in yeast strains. vtc3 and vtc3^TM1,mutant, vtc4 and vtc4^TM1,mutant, vtc1, vtc1^TM1,mutant and Vtc1^ΔN were complemented into the vtc2Δvtc3Δ, vtc2Δvtc4Δ, vtc2Δvtc1Δ background strains, respectively. Chemical shifts of vacuolar polyP are denoted. MDP was used as the std. reference. c smFRET analysis of VTC conformational changes. Top: Sites for fluorophores conjugation are highlighted in cyan on the molecular surface representation of InsP₆-activated VTC complex. The connection between TTM^Vtc4 and TM^Vtc4 (residues 472–618) is represented in magenta surface. The N-terminus of Vtc1_A protomer (N-ter, residues 1–21) is shown in cartoon representation. Bottom: smFRET profiles of VTC complex (Wt) in the presence and absence of 1 mM InsP₆ are colored in black and gray lines, respectively. smFRET profiles of VTC complex carrying Vtc1 N-terminus deletion (Vtc1^ΔN) in the presence and absence of 1 mM InsP₆ are colored in blue and green lines, respectively. Red dashed line indicates the position of FRET efficiency of 0.77.