Juxtaposition between endoplasmic reticulum (ER) and mitochondria is a common structural feature, providing the physical basis for intercommunication during Ca(2+) signalling; yet, the molecular mechanisms controlling this interaction are unknown. Here we show that mitofusin 2, a mitochondrial dynamin-related protein mutated in the inherited motor neuropathy Charcot-Marie-Tooth type IIa, is enriched at the ER-mitochondria interface. Ablation or silencing of mitofusin 2 in mouse embryonic fibroblasts and HeLa cells disrupts ER morphology and loosens ER-mitochondria interactions, thereby reducing the efficiency of mitochondrial Ca(2+) uptake in response to stimuli that generate inositol-1,4,5-trisphosphate. An in vitro assay as well as genetic and biochemical evidences support a model in which mitofusin 2 on the ER bridges the two organelles by engaging in homotypic and heterotypic complexes with mitofusin 1 or 2 on the surface of mitochondria. Thus, mitofusin 2 tethers ER to mitochondria, a juxtaposition required for efficient mitochondrial Ca(2+) uptake.

Mitofusin 2 tethers endoplasmic reticulum to mitochondria

This study shows that autophagosomes form at sites of contact between the endoplasmic reticulum and mitochondria, and that formation requires the SNARE protein syntaxin 17.

/pdf/autophagosomes-form-at-er-mitochondria-contact-sites-8xp82068qm.pdf

Autophagosomes form at ER–mitochondria contact sites

The ADP-ribosylation factor (ARF) small GTPases regulate vesicular traffic and organelle structure by recruiting coat proteins, regulating phospholipid metabolism and modulating the structure of actin at membrane surfaces. Recent advances in our understanding of the signalling pathways that are regulated by ARF1 and ARF6, two of the best characterized ARF proteins, provide a molecular context for ARF protein function in fundamental biological processes, such as secretion, endocytosis, phagocytosis, cytokinesis, cell adhesion and tumour-cell invasion.

ARF proteins: roles in membrane traffic and beyond.

The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER–mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are ∼10 nm at the smooth ER and ∼25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short “synthetic linker” (<5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.

https://rupress.org/jcb/article-pdf/174/7/915/1326212/915.pdf

Structural and functional features and significance of the physical linkage between ER and mitochondria

Communication between organelles is an important feature of all eukaryotic cells. To uncover components involved in mitochondria/endoplasmic reticulum (ER) junctions, we screened for mutants that could be complemented by a synthetic protein designed to artificially tether the two organelles. We identified the Mmm1/Mdm10/Mdm12/Mdm34 complex as a molecular tether between ER and mitochondria. The tethering complex was composed of proteins resident of both ER and mitochondria. With the use of genome-wide mapping of genetic interactions, we showed that the components of the tethering complex were functionally connected to phospholipid biosynthesis and calcium-signaling genes. In mutant cells, phospholipid biosynthesis was impaired. The tethering complex localized to discrete foci, suggesting that discrete sites of close apposition between ER and mitochondria facilitate interorganelle calcium and phospholipid exchange.

/pdf/an-er-mitochondria-tethering-complex-revealed-by-a-synthetic-4ihgwqmhmn.pdf

An ER-Mitochondria Tethering Complex Revealed by a Synthetic Biology Screen

The endoplasmic reticulum (ER) and mitochondria form contacts that support communication between these two organelles, including synthesis and transfer of lipids, and the exchange of calcium, which regulates ER chaperones, mitochondrial ATP production, and apoptosis. Despite the fundamental roles for ER–mitochondria contacts, little is known about the molecules that regulate them. Here we report the identification of a multifunctional sorting protein, PACS-2, that integrates ER–mitochondria communication, ER homeostasis, and apoptosis. PACS-2 controls the apposition of mitochondria with the ER, as depletion of PACS-2 causes BAP31-dependent mitochondria fragmentation and uncoupling from the ER. PACS-2 also controls formation of ER lipid-synthesizing centers found on mitochondria-associated membranes and ER homeostasis. However, in response to apoptotic inducers, PACS-2 translocates Bid to mitochondria, which initiates a sequence of events including the formation of mitochondrial truncated Bid, the release of cytochrome c, and the activation of caspase-3, thereby causing cell death. Together, our results identify PACS-2 as a novel sorting protein that links the ER–mitochondria axis to ER homeostasis and the control of cell fate, and provide new insights into Bid action.

/pdf/pacs-2-controls-endoplasmic-reticulum-mitochondria-1iqbo09hal.pdf

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

https://www.cell.com/cell/pdf/S0092-8674(02)01162-5.pdf

HIV-1 Nef Downregulates MHC-I by a PACS-1- and PI3K-Regulated ARF6 Endocytic Pathway

The final envelopment of herpesviruses during assembly of new virions is thought to occur by the budding of core viral particles into a late secretory pathway organelle, the trans-Golgi network (TGN), or an associated endosomal compartment. Several herpesvirus envelope glycoproteins have been previously shown to localize to the TGN when expressed independently from other viral proteins. In at least some cases this TGN localization has been shown to be dependent on clusters of acidic residues within their cytoplasmic domains. Similar acidic cluster motifs are found in endogenous membrane proteins that also localize to the TGN. These acidic cluster motifs interact with PACS-1, a connector protein that is required for the trafficking of proteins containing such motifs from endosomes to the TGN. We show here that PACS-1 interacts with the cytoplasmic domain of the HCMV envelope glycoprotein B (gB) and that PACS-1 function is required for normal TGN localization of HCMV gB. Furthermore, inhibition of PACS-1 activity in infected cells leads to a decrease in HCMV titer, whereas an increase in expression of functional PACS-1 leads to an increase in HCMV titer, suggesting that PACS-1 is required for efficient production of HCMV.

Role of PACS-1 in Trafficking of Human Cytomegalovirus Glycoprotein B and Virus Production

Erratum: PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis (EMBO Journal (2005) 24 (717-729) doi:10.1038/sj.emboj.7600559)

The Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded ORF50 protein is a potent transcriptional activator essential for triggering KSHV lytic reactivation. Despite extensive studies, little is known about whether ORF50 possesses the ability to repress gene expression or has an antagonistic action to cellular transcription factors. Previously, we demonstrated that human oncoprotein MDM2 can promote the degradation of ORF50 protein. Herein, we show that abundant ORF50 expression in cells can conversely downregulate MDM2 expression via repressing both the upstream (P1) and internal (P2) promoters of the MDM2 gene. Deletion analysis of the MDM2 P1 promoter revealed that there were two ORF50-dependent negative response elements located from −102 to −63 and from −39 to +1, which contain Sp1-binding sites. For the MDM2 P2 promoter, the ORF50-dependent negative response element was identified in the region from −110 to −25, which is coincident with the location of two known p53-binding sites. Importantly, we further demonstrated that overexpression of Sp1 or p53 in cells indeed upregulated MDM2 expression; however, coexpression with ORF50 protein remarkably reduced the Sp1- or p53-mediated MDM2 upregulation. Collectively, our findings propose a reciprocal negative regulation between ORF50 and MDM2 and uncover that ORF50 decreases MDM2 expression through repressing Sp1- and p53-mediated transactivation.

/pdf/kaposis-sarcoma-associated-herpesvirus-orf50-protein-1piw20t1.pdf

Chien Hui Hung

Papers

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

HIV-1 Nef Downregulates MHC-I by a PACS-1- and PI3K-Regulated ARF6 Endocytic Pathway

Role of PACS-1 in Trafficking of Human Cytomegalovirus Glycoprotein B and Virus Production

Erratum: PACS-2 controls endoplasmic reticulum-mitochondria communication and Bid-mediated apoptosis (EMBO Journal (2005) 24 (717-729) doi:10.1038/sj.emboj.7600559)

Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein Represses Cellular MDM2 Expression via Suppressing the Sp1- and p53-Mediated Transactivation