Figure EV4. The role of cap residues and cytoplasmic gate for SLC25A51 function.

• A
  Relative positions of cap residues and their interactions (dashed lines) in a cartoon representation of SLC25A51.
• B
  Free mitochondrial NAD⁺ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 12 biological replicates), wildtype ^FlagSLC25A51 (n = 12 biological replicates), and indicated mutants (n = 6 biological replicates). Measurements were taken at 24 h post‐transfection; the dashed line indicates the baseline defined by the empty vector control, and red denotes data equivalent to wildtype. Data are shown in box and whisker format, with hinges at 25^th and 75^th percentiles, whiskers represent min and max and the line is the median, ANOVA P < 0.0001, post‐hoc Dunnett's test compared to empty vector control (**) and compared to wildtype ^FlagSLC25A51 (#), **P < 0.01 and ^# P < 0.05. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype ^FlagSLC25A51 and the indicated variants using anti‐Flag Western blot; HSP60, loading control.
• C
  Relative position of the cytoplasmic gate in a cartoon representation of SLC25A51.
• D
  Time evolution of distance between indicated side chains for putative gate interactions. Interaction cutoff at 3.5 Å is shown as a dashed horizontal line.
• E
  Free mitochondrial NAD⁺ levels measured using a ratiometric biosensor in HeLa cells expressing empty vector control (n = 19 biological replicates), wildtype ^FlagSLC25A51 (n = 19 biological replicates), and indicated mutants (n = 4–7 biological replicates). Measurements taken at 24 h post‐transfection; empty vector control baseline is shown as dashed line and wildtype activity and equivalent is in red color. Data are shown in box and whisker format, with hinges at 25^th and 75^th percentiles, whiskers represent min and max and the line is the median, ANOVA P < 0.0001, post‐hoc Dunnett's test compared to empty vector control, ***P < 0.001. (bottom) Protein expression from HeLa cells transiently transfected with empty vector control, wildtype ^FlagSLC25A51 and the indicated variants using anti‐Flag Western blot; HSP60, loading control.
• F
  Quantitation of uptake of ³²P‐NAD⁺ after 1 h by E. coli cells expressing wildtype SLC25A51 (amino acids 29–297) and the indicated mutant; wildtype activity and equivalent is in red color and the wildtype baseline is shown as dashed line. Data are shown in box and whisker format, with hinges at 25^th and 75^th percentiles, whiskers represent min and max and the line is the median, n = 7 biological replicates, unpaired t‐test ***P < 0.001.
• G
  Representative phosphorimage of retained ³²P‐NAD⁺ after 1 h by E. coli cells expressing wildtype SLC25A51 (amino acids 29–297), indicated variant, or YFP control.
• H
  Membrane expression of wildtype SLC25A51 (amino acids 29–297), its indicated variant or YFP control in E. coli cells using anti‐SLC25A51 Western blot; TolC, loading control.

Source data are available online for this figure.