In this study, the subcellular localization and physiologic requirement for endogenous mammalian PNPase were determined. Our data showed that PNPase localized to mitochondria in multiple cell types, specifically to the IMS in a peripheral membrane protein complex. With a classical mitochondrial targeting sequence and prokaryotic and chloroplastic stroma counterparts, PNPase was expected to regulate mtRNA levels from within the matrix (35
). However, PNPase was surprisingly localized to the IMS, and mtRNAs were almost completely unaffected by PNPase reduction at its nadir. Mobilization of PNPase followed cytochrome c
release from mitochondria treated with proapoptotic stimuli, which strongly supported an IMS localization for PNPase and also suggested a potential route for interactions with cytosolic TCL1, which was how we initially identified PNPase (10
). An IMS location also could provide endogenous PNPase with RNA substrates under conditions that cause mitochondrial OM permeabilization (26
). In addition to its surprising IMS localization, PNPase exoribonuclease was unexpectedly required to maintain the filamentous structure of mitochondria, and its reduction caused a loss in Δψ with impairment of the respiratory chain, leading to a metabolic alteration that reduced cellular energy charge and caused decreased cell growth without increased cell death. Interestingly, more than 30 years ago an obscure RNase activity was isolated from the IMS of rat liver mitochondria (5
). Our identification of PNPase in the IMS may provide a source for this previously isolated RNase activity.
A variety of in vitro and in vivo systems in mammalian and yeast cells demonstrated and confirmed the IMS localization and membrane association of PNPase within mitochondria. Detailed import studies also showed that PNPase utilized a novel import pathway to reach its destination in the IMS (37
). Import required energized mitochondria and was coupled to the N-terminal processing of a 37-amino-acid mitochondrial targeting sequence (reference 37
and data not shown). We determined that basal and IFN-β-induced PNPase were imported into mitochondria, and appreciable nonmitochondrial PNPase was not detected under normal growth conditions. These results contrast with studies that show exogenous overexpression of PNPase decreases MYC
transcript levels and induces growth arrest and apoptosis, presumably from a cytosolic localization of PNPase (41
). Interestingly, high-level adenovirus-mediated PNPase overexpression (100 PFU) in these prior studies results in apoptosis, whereas a lower but still robust level of PNPase overexpression (25 PFU) does not produce enhanced apoptosis over control infections. We interpret these data with caution, because when combined with our current studies of endogenous PNPase localization they seem to suggest that growth arrest and apoptosis from exogenous PNPase overexpression could result from aberrant localization of PNPase, causing nonphysiologic activity in the cytosol. A cytosolic accumulation of PNPase could resemble a physiologic mobilization of PNPase from mitochondria, as demonstrated here for tBID-treated isolated mouse liver mitochondria and staurosporine- and Fas (data not shown)-treated HeLa cells. However, this mobilization follows cytochrome c
release, suggesting that physiologic PNPase mobilization does not induce apoptosis but rather may play a role in the processing of cytosolic RNAs during the clearance phase of cell death in vivo.
PNPase knockdown was correlated with mitochondrial dysfunction. Mitochondria became fragmented, which is a hallmark of mitochondrial dysfunction (7
). This result contrasted with a study that showed no change in the appearance or respiratory function of HeLa mitochondria with reduced PNPase expression (33
). A possible explanation for these discrepant results is that not enough time may have passed to permit the PNPase knockdown to become effective in these prior studies. We interpret the global dysfunction in respiration from PNPase deficiency as a consequence of the role for PNPase in mitochondrial homeostasis.
Based on our results, we propose a model for the range of cellular effects arising from a reduction in endogenous PNPase (Fig. ). Under physiologic growth conditions, PNPase localizes to the mitochondrial IMS as a peripheral membrane protein in a multimeric complex. A PNPase reduction of >65% results in a morphological change in the mitochondrial filamentous network, causing fragmentation and mitochondrial fission. This change is correlated with a loss of Δψ and a functional impairment of OX-PHOS, as observed with the reduction of the matrix-associated mtPAP RNA-processing protein (33
). However, reduced mtPAP decreases the steady-state level of multiple mtRNAs, suggesting that impaired OX-PHOS directly results from reduced expression of essential mtRNAs, which does not appear to be the source for OX-PHOS defects in PNPase-deficient mitochondria. Interestingly, an OX-PHOS impairment is likewise reported for the knockdown of AIF, a 57-kDa redox flavoprotein that also localizes as a peripheral IM protein in the IMS and might be expected, unlike PNPase, to affect respiration (43
). AIF is homologous to bacterial oxidoreductases, and knockout and RNAi knockdown reveal a dysfunction in complex I of the electron transport chain, resulting in elevated lactate and decreased ATP levels (48
). These metabolism endpoints are similar to the current findings for PNPase-deficient cells. However, PNPase deficiency does not alter the level of multiple mtRNAs, unlike mtPAP, nor does it appear to cause a defect in a specific respiratory chain complex, unlike AIF.
Mitochondrial dysfunction resulting from PNPase deficiency led to secondary effects including reduced ATP production. In addition to reduced ATP production from defective OX-PHOS, a complementary mechanism for further reducing ATP might include increased ATP catabolism as an adaptive response, through the ATP synthase acting as an ATP hydrolase, which would help stabilize Δψ and inhibit the onset of apoptosis (2
). Reduced cell energy charge, in turn, would enhance the phosphorylation of AMPK, which could slow cell growth through a potential link to p53-mediated inhibition of the cyclin E cell cycle checkpoint protein (21
). However, PNPase regulation of this cell growth pathway, of an alternative TOR-TSC2 growth pathway (19
), or of as-yet-undefined growth pathways is likely secondary to its primary role in mitochondrial homeostasis from its location within the IMS. Because of the complications imparted by delayed kinetics of PNPase knockdown, the generation of PNPase knockout cells will be critical for helping to determine the intricacies by which PNPase maintains mitochondrial integrity and respiratory function.