In Vivo Mitochondrial Import

Ni, Li; Heard, Thomas S.; Weiner, Henry

doi:10.1074/jbc.274.18.12685

Cited by 60 publications

(14 citation statements)

References 63 publications

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“…Sequence analysis of a variety of processed and unprocessed mitochondrial proteins shows that these proteins have an amino-terminal sequence rich in positive charges that also share an ability to form an amphiphilic ␣-helix (45). These observations have been confirmed experimentally (40,46,47).…”

Section: -E) No Mitochondrial Mrnas Destabilization Is Observed Aftesupporting

confidence: 62%

The 2–5A/RNase L/RNase L Inhibitor (RNI) Pathway Regulates Mitochondrial mRNAs Stability in Interferon α-treated H9 Cells

Roy¹,

Bisbal²,

Silhol³

et al. 2001

Journal of Biological Chemistry

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Interferon ␣ (IFN␣) belongs to a cytokine family that exhibits antiviral properties, immuno-modulating effects, and antiproliferative activity on normal and neoplasic cells in vitro and in vivo. IFN␣ exerts antitumor action by inducing direct cytotoxicity against tumor cells. This toxicity is at least partly due to induction of apoptosis. Although the molecular basis of the inhibition of cell growth by IFN␣ is only partially understood, there is a direct correlation between the sensitivity of cells to the antiproliferative action of IFN␣ and the down-regulation of their mitochondrial mRNAs. Here, we studied the role of the 2-5A/RNase L system and its inhibitor RLI in this regulation of the mitochondrial mRNAs by IFN␣. We found that a fraction of cellular RNase L and RLI is localized in the mitochondria. Thus, we down-regulated RNase L activity in human H9 cells by stably transfecting (i) RNase L antisense cDNA or (ii) RLI sense cDNA constructions. In contrast to control cells, no post-transcriptional down-regulation of mitochondrial mRNAs and no cell growth inhibition were observed after IFN␣ treatment in these transfectants. These results demonstrate that IFN␣ exerts its antiproliferative effect on H9 cells at least in part via the degradation of mitochondrial mRNAs by RNase L. Interferons (IFNs)1 belong to a family of cytokines produced and secreted by mammalian cells in response to various stimuli. Although originally discovered due to their participation in cellular defense against viral infection (1), IFNs are also known to modulate immune responses as well as control various cellular functions by altering the transcription of a large family of genes involved in cell proliferation and differentiation (2, 3).IFNs are negative regulators of cell proliferation through induction of cell cycle arrest and apoptosis, which has also been suggested to be of central importance in IFN antitumor action. IFN␣ (type I family) has emerged as a tumor suppressor protein. A number of clinical trials were thus carried out in cancer therapy with IFN␣ alone or in combination with other negative regulators of cell proliferation (4, 5). It has been shown to be effective against hematological malignancies including hairy cell leukemia, chronic myelogenous leukemia, and cutaneous T-cell lymphoma (6). IFN␣ has been used successfully in cancer therapy, but development of resistance to IFN␣ therapy has often been observed in patients. Although the molecular basis of the inhibition of cell growth by IFN␣ is partially understood, there is a correlation between the sensitivity of cells to the antiproliferative action of IFN␣ and the down-regulation of their mitochondrial mRNAs and the impairment of mitochondrial function upon IFN␣ treatment (7,8).In this work, we have studied the role of one of the enzymatic pathways induced by IFN␣, the 2-5A/RNase L pathway, in the regulation of mitochondrial mRNAs. The 2-5A/RNase L pathway is an RNA degradation pathway (9). IFN␣ induces the expression of 2-5A synthetase(s) which, upon activation by dou...

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Section: -E) No Mitochondrial Mrnas Destabilization Is Observed Aftesupporting

confidence: 62%

The 2–5A/RNase L/RNase L Inhibitor (RNI) Pathway Regulates Mitochondrial mRNAs Stability in Interferon α-treated H9 Cells

Roy¹,

Bisbal²,

Silhol³

et al. 2001

Journal of Biological Chemistry

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“…In the mitochondria we tested both the N and C-terminal orientation of a DD relative to Venus fluorescent protein, mDDn and mDDc. The mitochondrial DD reporter constructs contain the mitochondrial matrix targeting sequence from aldehyde dehydrogenase 2 (ALDH2, [18]). The ER DD fluorescent reporter, eDD, was made by fusing the secretion signal from Gaussia principis secreted luciferase (GLuc, [19]) to the N-terminus of DD-GFP.…”

Section: Resultsmentioning

confidence: 99%

Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain

Sellmyer

Chen²,

Egeler³

et al. 2012

PLoS ONE

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The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.

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“…Ni and colleagues 56 attached an MTS to the N-terminus of eGFP, and an ER targeting signal to its C-terminus. Analysis of the localization of the dual-signal eGFP in HeLa cells revealed it was present exclusively in mitochondria.…”

Section: Translated Proteinmentioning

confidence: 99%