Figure 6.
ORP2 is a major contributor to basal and SAC1mito-mediated PM PI4P turnover. (A) confocal section of a COS-7 cell over-expressing neonGreen-ORP2 for 24 h, showing a mainly cytosolic distribution. (B) as A, but the cell co-expresses mCherry VAPB, leading to partial localization of the neonGreen-ORP2 at the ER. (C) as A, but the cells were only transfected for 4 h before imaging, leading to lower levels of fluorescence more consistent with endogenous expression levels. (D) Cells were transfected with non-specific (ctrl) or ORP2 siRNA for 48h, and then expression of transiently transfected NeonGreen-ORP2, -ORP5 or EGFP-ORP8 was measured after an additional 24 h. P values from Šídák's multiple comparisons test are shown after a two-way repeated measures ANOVA (siRNA vs ORP, SS = 400018, DF = 2, MS = 200009, P < 0.005). (E)TIRFM images of COS-7 cells expressing EGFP-P4C PI4P biosensor and either control or an ORP2-directed siRNA pool. Cells were treated with 30 nM GSK-A1 at time 0. (F) Quantification of the experiment depicted in D. Data are grand means ± SEM of three experiments, quantifying 12 cells each. (G-H) as E-F, except cells were co-transfected with SAC1mito. Data in G are a grand means of three experiments, quantifying 10–12 cells each. (H) Area under the curve analysis of the data from F and H, with statistical analysis by two-way ANOVA. Pair-wise analysis gives the P values from a post hoc Šídák's multiple comparisons test. (I) Proposed model for ORP2 function in SAC1mito-mediated PM PI4P depletion; ORP2 removes PI4P from the PM, either tethered to VAPA or B at ER:PM contact sites (arrows 1 and 2) or untethered anywhere in the PM (arrow 3). From there, ORP2 diffuses to mitochondria to deliver PI4P for hydrolysis, either still tethered to VAPA or B (and thus occurring at ER:mitochondria contact sites, arrow 1) or untethered from VAPs (arrow 3).