Human telomerase reverse transcriptase (hTERT) is localized to mitochondria, as well as the nucleus, but details about its biology and function in the organelle remain largely unknown. Here we show, using multiple approaches, that mammalian TERT is mitochondrial, co-purifying with mitochondrial nucleoids and tRNAs. We demonstrate the canonical nuclear RNA [human telomerase RNA (hTR)] is not present in human mitochondria and not required for the mitochondrial effects of telomerase, which nevertheless rely on reverse transcriptase (RT) activity. Using RNA immunoprecipitations from whole cell and in organello, we show that hTERT binds various mitochondrial RNAs, suggesting that RT activity in the organelle is reconstituted with mitochondrial RNAs. In support of this conclusion, TERT drives first strand cDNA synthesis in vitro in the absence of hTR. Finally, we demonstrate that absence of hTERT specifically in mitochondria with maintenance of its nuclear function negatively impacts the organelle. Our data indicate that mitochondrial hTERT works as a hTR-independent reverse transcriptase, and highlight that nuclear and mitochondrial telomerases have different cellular functions. The implications of these findings to both the mitochondrial and telomerase fields are discussed.

Cytochrome P450 2E1 (CYP2E1) plays an important role in alcohol-induced toxicity and oxidative stress. Recently, we showed that this predominantly microsomal protein is also localized in rat hepatic mitochondria. In this report, we show that the N-terminal 30 amino acids of CYP2E1 contain a chimeric signal for bimodal targeting of the apoprotein to endoplasmic reticulum (ER) and mitochondria. We demonstrate that the cryptic mitochondrial targeting signal at sequence 21-31 of the protein is activated by cAMP-dependent phosphorylation at Ser-129. S129A mutation resulted in lower affinity for binding to cytoplasmic Hsp70, mitochondrial translocases (TOM40 and TIM44) and reduced mitochondrial import. S129A mutation, however, did not affect the extent of binding to the signal recognition particle and association with ER membrane translocator protein Sec61. Addition of saturating levels of signal recognition particle caused only a partial inhibition of CYP2E1 translation under in vitro conditions, and saturating levels of ER resulted only in partial membrane integration. cAMP enhanced the mitochondrial CYP2E1 (referred to as P450MT5) level but did not affect its level in the ER. Our results provide new insights on the mechanism of cAMP-mediated activation of a cryptic mitochondrial targeting signal and regulation of P450MT5 targeting to mitochondria.Accurate targeting of proteins to their designated subcellular compartments is critical for maintaining the distinctive structural and functional characteristics of individual cellular components. At least three major types of import/transport systems have been described for targeting proteins translated in the cytoplasm to different organelles in the eukaryotic cells. 1) Proteins destined for the ER, 1 Golgi, plasma membrane, and also those secreted out of cells, are targeted to the ER through a signal recognition particle (SRP)-dependent mechanism. This pathway is mostly co-translational and involves the delivery of nascent chains by SRP to the translocon complex on the ER membrane (1, 2). 2) Protein targeting to mitochondria occurs mostly by a post-translational mechanism, although exceptions to this generality have been reported (3, 4). As part of the mitochondrial targeting pathway, an unfolded polypeptide is brought in contact with the outer and inner membrane translocase complexes (TOM and TIM, respectively) (5). The protein is unidirectionally translocated through transmembrane protein channels formed of TOM40 and TIM23/TIM17 subunits, and its entry into the matrix space is finally facilitated by an ATP-dependent pull exerted by the mitochondrial Hsp70 chaperone protein (6).3) The peroxisomal protein targeting, although it occurs post-translationally, involves a distinct set of cytosolic receptors. These proteins not only guide the precursor proteins to the peroxisomal membrane receptor Pex, they also lead the polypeptide into the matrix compartment and eventually recycle back to the cytosol for reutilization (7, 8). As predicted by the signal hypothesis, the targe...

The Arabidopsis HY4 gene, required for bluelight-induced inhibition of hypocotyl elongation, encodes a 75-kDa flavoprotein (CRY1) with characteristics of a bluelight photoreceptor. To investigate the mechanism by which this photoreceptor mediates blue-light responses in vivo, we have expressed the Arabidopsis HY4 gene in transgenic tobacco. The transgenic plants exhibited a short-hypocotyl phenotype under blue, UV-A, and green light, whereas they showed no difference from the wild-type plant under red/farred light or in the dark This phenotype was found to cosegregate with overexpression of the HY4 transgene and to be fluence dependent. We concluded that the short-hypocotyl phenotype of transgenic tobacco plants was due to hypersensitivity to blue, UV-A, and green light, resulting from overexpression of the photoreceptor. These observations are consistent with the broad action spectrum for responses mediated by this cryptochrome in Arabidopsis and indicate that the machinery for signal transduction required by the CRYI protein is conserved among different plant species. Furthermore, the level of these photoresponses is seen to be determined by the cellular concentration of this photoreceptor.Light from the blue and near-UV spectral regions has profound effects on plant growth and development. Some prominent examples of this are the photomorphogenesis and photomovement responses, including phototropism, chloroplast rearrangement, stomatal opening, and inhibition of hypocotyl elongation (1-7). In Arabidopsis thaliana, the hypocotylelongation response is mediated by at least two photoreceptors: the red/far-red photoreceptor, phytochrome, and the blue/UV-A photoreceptor, cryptochrome (8-10). In spite of the fact that plant responses to blue light were recognized over a century ago (11), our understanding of the blue-light photoreceptor is very limited in comparison with that of phytochrome. We have previously demonstrated that the Arabidopsis RY4 gene, required for blue-light-dependent inhibition of hypocotyl elongation (8), contained an open reading frame encoding a protein with significant sequence similarity to microbial DNA photolyase (12). As photolyase is a rare class of flavoenzyme that functions as the result of photon absorption, we proposed that the protein encoded by HY4 was a flavin-type blue-light photoreceptor (12). We have recently demonstrated that the HY4 gene product was indeed a flavoprotein (13). We refer to this protein as CRY1, after cryptochrome, the name commonly given to plant blue/UV-A light photoreceptors.Further understanding of CRY1 and its function in bluelight signal transduction will require the development of assays to enable us to explore the relationship between structure and biochemical and physiological properties of this photoreceptor. One such system involves transgenic overexpression studies, which have significantly improved our understanding of the plant photoreceptor phytochrome (14-26). In these studies, it was shown that phytochrome-overexpressing transgenic plant...