A high-throughput screening for mammalian cell death effectors identifies the mitochondrial phosphate carrier as a regulator of cytochrome c release

Abstract

Functional annotation of complex genomes requires the development of novel experimental platforms with increased capacity. Here, we describe a high-throughput system designed to identify cDNAs whose overexpression induces morphologically distinct cell death modalities. The methodology incorporates two robotized steps, and relies on coexpression of library clones with GFP to reveal the morphological features presented by the dying cells. By using this system we screened 135 000 cDNA clones and obtained 90 independent molecules. Interestingly, three death categories were identified, namely; apoptotic, vacuolated and autophagic. Among the pro-apoptotic clones, we found four members of the mitochondrial carrier family: the phosphate and adenine nucleotide (type 3) transporters, and the mitochondrial carrier homologs (MTCHs) 1 and 2. Expression of these molecules induced cytochrome c release and caspase-9-dependent death. One of them, the phosphate carrier, was able to interact with members of the permeability transition pore complex ANT1 and VDAC1, and its binding to ANT1 was stabilized in the presence of apoptotic activators. Depletion of this carrier by siRNA delayed cytochrome c mobilization and apoptosis. These results attribute a previously undescribed apoptotic function to the phosphate carrier and, more generally, suggest that a common property of various mitochondrial transporters was exploited during evolution to regulate apoptosis.

Role of the c subunit of the FOATP synthase in mitochondrial permeability transition

“…The mitochondrial F 1 F O ATP synthase has previously been shown to interact with ANT and the mitochondrial phosphate carrier (SLC25A3), both of which are involved in MPT, 29,30 to form multiprotein complexes that catalyze the final steps of mitochondrial ATP synthesis also known as "ATP synthasomes." 31 In addition, CYPD as well as two distinct anti-apoptotic members of the BCL-2 family, namely BCL-X L and MCL-1, have recently been reported to regulate mitochondrial ATP synthesis by physically interacting with the F 1 F O ATP synthase.…”

“…By ion exchange chromatography, we have succeeded in separating a multiprotein complex that contains ANT, VDAC1, CYPD, hexokinase, the F O c subunit as well as multiple F 1 components (data not shown), in line with the existence of a supramolecular entity involving several constituents of the PTPC as well as the ATP synthasome. 27,[29][30][31] Now, it will be interesting to identify the direct physical interactors of the F O c subunit among the core PTPC components and its best known regulators (including pro-and anti-apoptotic BCL-2 family members as well as the oncosuppressor protein TP53). 63,64 To test the actual pathophysiological relevance of our findings, it will be crucial to generate appropriate knockout models.…”

Adenine nucleotide translocase: a component of the phylogenetically conserved cell death machinery

“…48 The mitochondrial phosphate carrier (SLC25A3), another member of the MCP family, has been found to interact with the viral Bcl-2 analog vMIA (encoded by the genome of Cytomegalovirus) 49 and to induce apoptosis on overexpression. 50 Accordingly, knockdown of this protein, which can interact with ANT1 and with the voltage-dependent anion channel (VDAC), limited staurosporine (STS)-induced cytochrome c release and apoptosis. 50 In the same context (a highthroughput screening for mammalian cell death effectors), 50 also ANT3 and the mitochondrial carrier homologs (MTCHs) 1 and 2 were identified.…”

Unexpected role of the phosphate carrier in mitochondrial fragmentation

“…A genome-wide cDNA screen identified PiC as one of the few proteins that, if overexpressed, is able to efficiently trigger the intrinsic pathway of apoptosis. 7 PiC was also found to functionally interact with the cytomegalovirus (CMV)-encoded protein vMIA (i.e. viral mitochondrial inhibitor of apoptosis).…”