Fig. 5: Phospholipid transport across the OMM is slowed more than 10-fold in yeast mitochondria lacking VDAC homologs.

a Assay schematic. NBD-PS (indicated PS) is added to yeast mitochondria. After insertion into the outer leaflet of the OMM, NBD-PS flips reversibly across the membrane where it encounters IMM-localized PS decarboxylase (Psd1) which converts it to NBD-PE (indicated PE). Psd1 can act in trans, hydrolyzing PS in the inner leaflet of the OMM as shown. It can also act in cis on NBD-PS molecules that are delivered to the IMS side of the IMM. The decarboxylation of exogenously supplied NBD-PS can be described using a 4-state kinetic model and 5 effective rate constants as shown: deposition of NBD-PS into the OMM (k₀), desorption from the OMM (k₁), scrambling across the OMM (k₂ and k₃, presumed to be identical and written as k_2/3), and irreversible conversion to NBD-PE (k₄). b Thin layer chromatogram, visualized with a ChemiDoc fluorescence imager, of a decarboxylation assay time-course using mitochondria from wild-type yeast (top panel) and the por1∆por2∆ double mutant (bottom panel, labeled ∆∆). This assay was performed 5 times with similar results. c Time courses of NBD-PS decarboxylation corresponding to (b) (the traces are as in Fig. S12, from 2 biological replicates, with at least 2 technical replicates per assay). The data for wild-type mitochondria were analyzed using a 4-state kinetic model (panel a) with k₀ = 1 s⁻¹, k₁ = 0.05 s⁻¹, and k₂ = k₃ > 0.01 s⁻¹ to obtain k_2/3 and k₄. The fitting yielded k₄ = 0.0018 s⁻¹. Data for the por1∆por2∆ double mutant were analyzed using the same kinetic model with k₀ = 1 s⁻¹, k₁ = 0.05 s⁻¹ and k₄ = 0.0018 s⁻¹ to determine k_2/3. Example time courses of the 4-state model P₃ pool using wild-type (black) or porin mutant (green) rate constants are shown with circles representing experimental data (see Methods for details of the model). The data are representative of assays done on two biological replicates with at least 2 technical replicates per assay. d Best fitting scrambling rates (k_2/3) for wild-type and por1∆por2∆ mitochondria across several experiments obtained as in panel c. The corresponding half-times are 25 and 265 s for wild-type and por1∆por2∆ mitochondria, respectively. **p = 0.00738 (2-tailed (unpaired) T test). Error bars correspond to SEM.