PACS-2 controls endoplasmic reticulum–mitochondria communication and Bid-mediated apoptosis

Simmen, Thomas; Aslan, Joseph E.; Blagoveshchenskaya, Anastassia D.; Thomas, Laurel; Wan, Lei; Xiang, Yang; Féliciangéli, Sylvain; Hung, Chien Hui; Crump, Colin M.; Thomas, Gary

doi:10.1038/sj.emboj.7600559

Cited by 479 publications

(432 citation statements)

References 45 publications

(83 reference statements)

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“…Thus, ER oxidative protein folding appears to determine the properties of the MAM, a function that may explain the increased apposition between the ER and mitochondria upon ER stress (20). In line with these findings, a disruption of the MAM results in a reduced folding capacity of the ER, concomitant with ER stress (12,21,22).…”

supporting

confidence: 72%

“…Membrane Fractionation, Protein-Protein Binding, and Biotinylation Assays-Mitochondria and light membranes were separated as described previously (12). The MAM OptiPrep TM gradient protocol was described previously (2).…”

Section: Methodsmentioning

confidence: 99%

“…Insight into how the ER establishes its domains would, however, help our understanding of the functioning of the MAM and other domains of the ER. Our laboratory has so far identified one such mechanism in the form of the cytosolic phosphofurin acidic cluster sorting protein 2 (PACS-2) (2,12). PACS-2, like other MAM targeting factors to be characterized, influences the properties of the MAM by regulating its composition and impacts on MAM formation as well.…”

mentioning

confidence: 99%

“…PACS-2, like other MAM targeting factors to be characterized, influences the properties of the MAM by regulating its composition and impacts on MAM formation as well. Through these functions, PACS-2 is an important regulator of apoptosis onset (12,26,27). …”

mentioning

confidence: 99%

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Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

Bui

Gilady

Fitzsimmons

et al. 2010

Journal of Biological Chemistry

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144

143

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The mitochondria-associated membrane (MAM) has emerged as an endoplasmic reticulum (ER) signaling hub that accommodates ER chaperones, including the lectin calnexin. At the MAM, these chaperones control ER homeostasis but also play a role in the onset of ER stress-mediated apoptosis, likely through the modulation of ER calcium signaling. These opposing roles of MAM-localized chaperones suggest the existence of mechanisms that regulate the composition and the properties of ER membrane domains. Our results now show that the GTPase Rab32 localizes to the ER and mitochondria, and we identify this protein as a regulator of MAM properties. Consistent with such a role, Rab32 modulates ER calcium handling and disrupts the specific enrichment of calnexin on the MAM, while not affecting the ER distribution of protein-disulfide isomerase and mitofusin-2. Furthermore, Rab32 determines the targeting of PKA to mitochondrial and ER membranes and through its overexpression or inactivation increases the phosphorylation of Bad and of Drp1. Through a combination of its functions as a PKA-anchoring protein and a regulator of MAM properties, the activity and expression level of Rab32 determine the speed of apoptosis onset.

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supporting

confidence: 72%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

Bui

Gilady

Fitzsimmons

et al. 2010

Journal of Biological Chemistry

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144

143

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“…In fact, it was shown that mitochondrial ROS production could directly control cell surface protein expression (37). In addition, knockdown of PACS-2 expression not only uncouples the ER from mitochondria but also induces a misfolded protein response in the ER, confirming that MAM has a direct impact on ER oxidative protein folding (38).…”

Section: Gtgcccgccatgggcttcacgctggcgcccagcctggggtgcttcgtgggctcccgctttmentioning

confidence: 66%

The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway

Zhou

Dang

Zheng

2014

J Virol

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The HIV-1 Env glycoprotein is folded in the endoplasmic reticulum (ER), which is necessary for viral entry and replication. Currently, it is still unclear how this process is regulated. The glycoprotein folding in the ER is controlled by the ER-associated protein degradation (ERAD) pathway, which specifically targets misfolded proteins for degradation. Previously, we reported that HIV-1 replication is restricted in the human CD4 ؉ T cell line CEM.NKR (NKR). To understand this mechanism, we first analyzed cellular protein expression in NKR cells and discovered that levels of the mitochondrial translocator protein TSPO were upregulated by ϳ64-fold. Notably, when NKR cells were treated with TSPO antagonist PK-11195, Ro5-4864, or diazepam, HIV restriction was completely disrupted, and TSPO knockdown by short hairpin RNAs (shRNAs) achieved a similar effect. We next analyzed viral protein expression, and, interestingly, we discovered that Env expression was specifically inhibited. Both TSPO knockdown and treatment with TSPO antagonist could restore Env expression in NKR cells. We further discovered that Env proteins were rapidly degraded and that kifunensine, an ERAD pathway inhibitor, could restore Env expression and viral replication, indicating that Env proteins were misfolded and degraded through the ERAD pathway in NKR cells. We also knocked out the TSPO gene in 293T cells using CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeat [CRISPR]/CRISPRassociated-9) technology and found that TSPO could similarly inhibit Env expression in these cells. Taken together, these results demonstrate that TSPO inhibits Env protein expression through the ERAD pathway and suggest that mitochondria play an important role in regulating the Env folding process. IMPORTANCEThe HIV-1 Env glycoprotein is absolutely required for viral infection, and an understanding of its expression pathway in infected cells will identify new targets for antiretroviral therapies. Env proteins are folded in the ER and secreted through the classical secretory pathway. The Env folding process involves extensive cross-linking of 10 Cys residues by disulfide bond formation and heavy N-glycosylation on ϳ30 Asn residues. Currently, it is still unclear how this process is regulated. Here, we studied this mechanism in the HIV nonpermissive human CD4 ؉ T cell line CEM.NKR. We found that Env proteins were rapidly degraded through a cellular pathway that specifically targets misfolded proteins, resulting in inhibition of Env expression. Importantly, we have identified a mitochondrial translocator protein, TSPO, which could trigger this degradation by interfering with the Env folding process. Further characterization of TSPO antiviral activity will reveal a novel antiretroviral mechanism that targets the Env protein.

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Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

2007

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Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub‐domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria‐associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing the substantial protein yields from HCMV‐infected primary fibroblasts and from transfected HeLa cells. Moreover, this unit includes a protocol that allows visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. Curr. Protoc. Cell Biol. 37:3.27.1‐3.27.23. © 2007 by John Wiley & Sons, Inc.

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PACS-2 controls endoplasmic reticulum–mitochondria communication and Bid-mediated apoptosis

Cited by 479 publications

References 45 publications

Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

Rab32 Modulates Apoptosis Onset and Mitochondria-associated Membrane (MAM) Properties

The Mitochondrial Translocator Protein, TSPO, Inhibits HIV-1 Envelope Glycoprotein Biosynthesis via the Endoplasmic Reticulum-Associated Protein Degradation Pathway

Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

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