Polynucleotide phosphorylases (PNPases) encompass an evolutionarily conserved enzyme family whose members regulate RNA levels in bacteria and plants. In Escherichia coli
, PNPase associates with an RNA helicase and enolase in a degradosome complex, where it functions as both an exonuclease and a poly(A) polymerase to control RNA stability (32
). In plants, PNPase localizes to the chloroplast stroma, where it performs polyadenylation and exonucleolytic degradation to mediate RNA turnover (3
). In both bacteria and plants, poly(A) addition to RNA constitutes a signal for PNPase-mediated RNA degradation.
Interestingly, PNPase is absent from archaea and single-cell eukaryotes including Saccharomyces cerevisiae
, Schizosaccharomyces pombe
, and Plasmodium falciparum
but is present in animals including the worm, fly, mouse, and human (24
). This odd phylogenetic distribution suggests that PNPase might possess a unique function in animals, rather than its evolutionarily conserved role in the degradosome, as shown for prokaryotes and chloroplasts.
Recently, human PNPase was identified as an up-regulated gene in terminally differentiated melanoma cells and in senescent progeroid fibroblasts, implying a potential role in growth inhibition (25
). Human PNPase is a 85-kDa nucleus-encoded protein that localizes to the mitochondrion (4
) and functions in vitro as a phosphate-dependent exoribonuclease (8
) and poly(A) polymerase (33
). Analogous to bacterial and chloroplastic PNPase, mammalian PNPase could be expected to regulate RNA levels in the mitochondrial matrix. However, robust PNPase overexpression inhibits cell growth and induces apoptosis with down-regulation of MYC
), suggesting that PNPase may degrade mRNAs in the cytosol. These observations deviate from a predicted role for PNPase in regulating mitochondrial RNA levels (33
). A possible explanation for this disparity includes aberrant targeting of exogenous PNPase to the cytosol. Alternatively, PNPase might function within and outside mitochondria, akin to cytochrome c
, apoptosis-inducing factor, Smac/DIABLO, HtrA2/Omi, Bit1, and endonuclease G, with the intermembrane space providing a location for mitochondrial activity and conditions that cause outer membrane permeability enabling cytosolic access (41
). Indeed, we have identified PNPase by its interaction with the cytosolic TCL1 oncoprotein in recent work (8
). Furthermore, our accompanying paper shows that PNPase does not regulate mitochondrial RNA levels but, after treatment with apoptotic stimuli, is mobilized late after cytochrome c
Given that the specific localization of PNPase is not understood, we have characterized the PNPase biogenesis pathway in detail. Our accompanying study shows that endogenous PNPase resides in the intermembrane space in mouse liver mitochondria and, when heterologously expressed in S. cerevisiae
, localizes in mitochondria and not the cytosol (4
). Moreover, PNPase has a typical N-terminal targeting sequence that confers mitochondrial localization.
Mitochondria possess an elaborate set of translocons on the outer membrane (TOM) and translocons on the inner membrane (TIM) to mediate the import and assembly of nucleus-coded proteins such as PNPase (20
). Whereas the TIM22 complex mediates the import of inner membrane proteins, including the carrier family and the import component Tim22, the TIM23 translocon imports proteins with a typical N-terminal targeting sequence (21
). After passing through the TOM, the precursor engages the TIM23 complex; translocation initiation into the TIM23 complex requires the electrochemical membrane potential (Δψ) of the inner membrane. For proteins targeted to the matrix, the Hsp70 motor mediates translocation. The matrix processing protease (MPP), consisting of the heterodimer Mas1/Mas2, typically mediates cleavage of the presequence (9
). Precursors destined for the intermembrane space, such as cytochromes b2
, contain a bipartite targeting sequence. The N terminus directs the precursor to TIM23. After cleavage by the MPP, the precursor arrests at the “stop-transfer” domain in the translocon (12
). The inner membrane protease (IMP), consisting of the complex Imp1/Imp2 and facing the intermembrane space, mediates a second cleavage, thereby trapping cytochrome b2
in the intermembrane space (9
Additional proteases in the mitochondrial inner membrane have been implicated in import pathways in recent studies (2
). Beyond their role in protein degradation, the m-
AAA (ATPases associated with a number of diverse cellular activities) proteases Yta10 and Yta12 are required for maturation of the ribosomal protein MrpL32 and cytochrome c
peroxidase (Ccp1) (7
). The evolutionarily conserved rhomboid protease processes the substrates Mgm1, involved in mitochondrial dynamics, and Ccp1 (7
). These recent studies confirm that the mitochondrial proteolytic system is crucial for the maturation and assembly of precursors in the mitochondrion.
Import mechanisms into the intermembrane space are diverse (17
). Translocation of proteins such as cytochromes b2
requires the hydrolysis of matrix ATP and Δψ. For proteins that lack a typical N-terminal targeting sequence and do not require a Δψ for translocation (thereby bypassing the TIM), import mechanisms rely on (i) the energy gain of protein folding, often around a cofactor, or (ii) the association of a protein with a high-affinity binding site, such as another protein.
Here we use the conserved import machinery of S. cerevisiae
to investigate the pathway by which PNPase is localized to the intermembrane space. Import depends on the presence of Δψ, and the presequence is cleaved by MPP. Translocation of PNPase into the intermembrane space requires the i
-AAA protease Yme1, which possesses chaperone-like activity (23
); this constitutes the first evidence of a translocation function for this protease, presenting a new twist on biogenesis mechanisms in the intermembrane space. Conceivably, other intermembrane space proteins, particularly those involved in the execution events of apoptosis, may also utilize Yme1 for import.