Increasing concentrations of digitonin selectively permeabilized outer and inner mitochondrial membranes and ARL2 staining was seen only after permeabilization of the inner mitochondrial membrane, the same condition required to visualize HSP60, a well-documented matrix protein. In contrast, cytochrome c staining of mitochondria was seen at lower concentrations of digitonin. The digitonin permeabilization approach does not rule out the presence of ARL2 in the IMS if that pool of the GTPase is not fixed well by paraformaldehyde. Thus, we conclude that a pool of mitochondria-associated ARL2 is in the matrix, though we cannot exclude the possibility that some ARL2 is also in the IMS. Indeed, we believe there is a pool of ARL2 in the IMS, based upon our earlier sub-mitochondria fractionation data and more recent techniques that combine proteomics with spatially restricted protein tagging ; each of which concluded that ARL2 is in the IMS. Because ARL2 is present in mitochondria in amounts that preclude a stoichiometric binding to any of the TG100713 complexes of the electron transport chain or transporters and is not found in any stable complexes, it is likely that ARL2 actions in the matrix are catalytic or regulatory and transient in nature, rather than as a stoichiometric component of a complex. How then does the depletion of ARL2 cause these mitochondrial Salicylanilide defects? We believe the best model is that ARL2 is involved in remodeling of the inner mitochondrial membrane, likely at crista junctions. Knockdown of either of the inner membrane proteins ChChd3 or ChChd6 results in mitochondrial fragmentation, perinuclear clustering, and loss of cell proliferation and ATP levels. ChChd3 siRNA also decreased cell proliferation without increasing apoptosis, again similar to ARL2 siRNA. The two ChChd proteins bind to each other and to Mitofilin, a core component of the MINOS or MitOS complex that is key to crista junction formation and stabilization. The model for ARL2 affecting inner membrane remodeling also derives from analogy to the actions of the ARF proteins, which are intimately involved in vesicle biogenesis and membrane remodeling at the Golgi and endosomes.