Fig. 4. Interaction of PDZD8 promotes GLS1 activity.

a, b PDZD8-CT that constitutively interacts with GLS1, promotes GLS1 activity in vitro independently of AMPK. Recombinant KGA (left panel) or GAC (right panel) isozyme of GLS1 was mixed with recombinant PDZD8-CT, followed by determining the enzymatic activities of GLS1 in the presence (b) or absence (a) of 20 mM K₂HPO₄ (Pi). Data are shown as mean ± SD; n = 3 for each condition. See also K_m and k_cat values for each reaction in Supplementary information, Table S2. The experiments in (a) and Fig. 3c were performed at the same time and shared control (the KGA- and GAC-alone groups), and ditto for b and Fig. 3f. c, d PDZD8-CT promotes glutaminolysis and OCR in high glucose. PDZD8^–/– MEFs were infected with lentiviruses carrying full-length (FL) PDZD8 or PDZD8-CT, followed by incubating in a medium containing doxycycline for 12 h. Cells were then labeled with [U-¹³C]-glutamine to determine glutaminolysis (c, performed as in Fig. 1a) or subjected to Seahorse analyzer to determine OCR (d). Data are shown as mean ± SEM; n = 4 (c), or labeled on the panel (d; representing biological replicates) for each condition; P values were determined by two-way ANOVA, followed by Tukey (P values in c represent the comparisons between the starved and the unstarved groups of each genotype). e AMPK releases the autoinhibition of PDZD8-NT towards PDZD8-CT. MEFs stably expressing FLAG-tagged PDZD8-FL or PDZD8-CT were glucose-starved for 2 h, followed by immunoprecipitation with anti-FLAG and immunoblotting for co-precipitated GLS1. f AMPK causes PDZD8-NT to move away from PDZD8-CT. AMPKα^–/– MEFs (middle panel), or PDZD8^–/– MEFs (right panel) were infected with lentiviruses carrying RFP-PDZD8-GFP (middle and right panels) or RFP-PDZD8-T527A-GFP (right panel), followed by determination of the fluorescence lifetime of GFP (FRET donor; see principles of this assay on the left panel). Data are shown as mean ± SEM; n values were labeled on the panel representing cell numbers; P values were determined by two-way ANOVA, followed by Tukey. g–j GLS1-33A that loses the interface for PDZD8 fails to promote GLS1 activity (g, h), glutaminolysis (i), or OCR (j) in low glucose. Experiments in g and h were performed as in a and b, except that the recombinant KGA-33A (left panel) and GAC-33A (right panel) were mixed with AMPK-phosphorylated PDZD8. See also lowered K_m and increased k_cat values in each reaction in Supplementary information, Table S2. Experiments in i and j were performed as in c and d, except that GLS1^–/– MEFs with WT KGA or KGA-33A stably expressed were used. Data are mean ± SD; n = 3 (g, h) or 4 (i), or labeled on the panel (j; representing biological replicates) for each condition; P values were determined by two-way ANOVA, followed by Tukey (i) or by unpaired two-tailed Student’s t-test (j). Experiments in this figure were performed three times.