Fig. 4. Localization and characterization of FSP1 as vitamin K reductase.

a Fluorescence confocal microscope imaging of FSP1-sfGFP fusion (green) with cell organelle markers (red) of ER, mitochondrion, and Golgi. FSP1-sfGFP and the cell organelle marker were transiently co-transfected into HEK293 cells for 48 h. b Effect of FSP1 myristylation site mutation (G2A) and mutations of residue responsible for the dinucleotides (NDAH and FAD) binding on VKR activity. Wild-type FSP1 or its mutants was transiently expressed in TKO cells for VKR activity assay. Wild-type FSP1 activity was normalized to 100% (indicated by dotted line). Bottom: Immunoblotting of FSP1, its mutants, and GAPDH (loading control) in the corresponding cells. c AlphaFold model of FSP1 (gray) with FAD (cyan), NADH (gold) and vitamin K (green) based on ligand positions in the yeast NDH-2 enzyme Ndi1 (PDBcode 4G73). The GxGxxG motifs are colored purple (Gly18-Gly23) and orange (Gly149-Gly154) corresponding to interactions with the nucleotide motifs of FAD and NADH respectively. Residues Asp41 and His174 located near the ribose hydroxyls from the two nucleotides are colored pink as is Phe21 from the first GxGxxG region. d FSP1 residues surrounding the isoalloxazine of FAD, nicotinamide of NADH and quinone of vitamin K in the model that were mutated in this study are colored pink. e Effect on FSP1 activity due to mutations surrounding the proposed vitamin K binding site. VKR activity was determined as described above (Fig. 4b). Bottom: Immunoblotting of FSP1, its mutants, and GAPDH (loading control) in the corresponding cells. f Proposed activation mechanism of the activity-based fluorescent probe of vitamin K (VK-ASM) towards FSP1. The probe is inactive until the vitamin K moiety is reduced to hydroquinone, and the rearrangement of the reduced intermediated releases the strong fluorescent tag. g Effect of FSP1 mutations on the reduction of vitamin K and CoQ₁₀. Wild-type FSP1 and its mutants were transiently expressed in TKO cells for forty-eight hours, cell lysate was used for activity assay using the activity-based fluorescent probe of vitamin K (VK-ASM) and CoQ₁₀ (NIR-ASM) as the substrate. Fluorescence intensity of the wild-type FSP1 for each probe was normalized to 100% (indicated by the dotted line). Data are presented as mean ± SD of three independent experiments (n = 3) in Fig. 4b, e, g. Similar results were observed at least twice as shown in Fig. 4b, e.