The oxysterol‐binding protein (OSBP)‐related proteins ORP5 and ORP8 have been shown recently to transport phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) at ...ER–PM contact sites. PS is also transferred from the ER to mitochondria where it acts as precursor for mitochondrial PE synthesis. Here, we show that, in addition to ER–PM contact sites, ORP5 and ORP8 are also localized to ER–mitochondria contacts and interact with the outer mitochondrial membrane protein PTPIP51. A functional lipid transfer (ORD) domain was required for this localization. Interestingly, ORP5 and ORP8 depletion leads to defects in mitochondria morphology and respiratory function.
Synopsis
This study provides evidence for a novel localization of the PS lipid transfer proteins ORP5 and ORP8 at ER–mitochondria contact sites, in addition to ER–PM contacts. Depletion of ORP5 and ORP8 leads to defects in mitochondria morphology and respiratory function.
In addition to ER–PM contact sites, ORP5 and ORP8 also localize at ER–mitochondria contacts.
ORP5/ORP8 interacts with the mitochondrial protein PTPIP51.
ORP5/ORP8 targeting to ER–mitochondria contact sites and interaction with mitochondrial protein PTPIP51 depends on their lipid binding/transfer ability.
ORP5/ORP8 is involved in the maintenance of mitochondria morphology and respiratory function.
This study provides evidence for a novel localization of the PS lipid transfer proteins ORP5 and ORP8 at ER–mitochondria contact sites, in addition to ER–PM contacts. Depletion of ORP5 and ORP8 leads to defects in mitochondria morphology and respiratory function.
In all eukaryotic cells, the endoplasmic reticulum (ER) and the mitochondria establish a tight interplay, which is structurally and functionally modulated through a proteinaceous tether formed at ...specific subdomains of the ER membrane, designated mitochondria-associated membranes or MAMs. The tethering function of the MAMs allows the regulation of lipid synthesis and rapid transmission of calcium (Ca2+) signals between the ER and mitochondria, which is crucial to shape intracellular Ca2+ signaling and regulate mitochondrial bioenergetics. Research on the molecular characterization and function of MAMs has boomed in the last few years and the list of signaling and structural proteins dynamically associated with the ER–mitochondria contact sites in physiological and pathological conditions, is rapidly increasing along with the realization of an unprecedented complexity underlying the functional role of MAMs. Besides their established role as a signaling hub for Ca2+ and lipid transfer between ER and mitochondria, MAMs have been recently shown to regulate mitochondrial shape and motility, energy metabolism and redox status and to be central to the modulation of various key processes like ER stress, autophagy and inflammasome signaling. In this review we will discuss some emerging cell-autonomous and cell non-autonomous roles of the MAMs in mammalian cells and their relevance for important human diseases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.
•Detailed description of new MAM functions in cell signaling•Involvement of the MAMs in endoplasmic reticulum stress•Description of the link of MAMs with inflammatory responses•Implication of MAM dysfunction in neurodegenerative disorders•Linking MAM dysfunction with cancer and cancer therapy
The Golgi as an Assembly Line to the Autophagosome De Tito, Stefano; Hervás, Javier H.; van Vliet, Alexander R. ...
Trends in biochemical sciences,
June 2020, 2020-06-00, 20200601, Letnik:
45, Številka:
6
Journal Article
Recenzirano
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Autophagy is traditionally depicted as a signaling cascade that culminates in the formation of an autophagosome that degrades cellular cargo. However, recent studies have identified myriad pathways ...and cellular organelles underlying the autophagy process, be it as signaling platforms or through the contribution of proteins and lipids. The Golgi complex is recognized as being a central transport hub in the cell, with a critical role in endocytic trafficking and endoplasmic reticulum (ER) to plasma membrane (PM) transport. However, the Golgi is also an important site of key autophagy regulators, including the protein autophagy-related (ATG)-9A and the lipid, phosphatidylinositol-4-phosphate PI(4)P. In this review, we highlight the central function of this organelle in autophagy as a transport hub supplying various components of autophagosome formation.
The Golgi complex regulates production and delivery of proteins and lipids, and is a site of lipid metabolism needed for autophagy, in particular PI(4)P.ATG9A is the sole transmembrane ATG protein and has a crucial role in the formation of the autophagosome, one new role being the delivery of the metabolizing enzymes of PI to the nascent autophagosome.ATG9A trafficking from the Golgi and recycling endosome is controlled by the coat adaptor complexes AP1, AP2, and AP4, and several BAR-domain containing proteins BIF1, SNX18, and recently Arfaptin2.The control of ATG9A delivery to the forming autophagosome allows in situ PI(4)P production for the initiation of phagophore formation.
The integrity of ER-mitochondria appositions ensures transfer of ions and phospholipids (PLs) between these organelles and exerts crucial effects on mitochondrial bioenergetics. Malfunctions within ...the ER-mitochondria contacts altering lipid trafficking homeostasis manifest in diverse pathologies, but the molecular effectors governing this process remain ill-defined. Here, we report that PERK promotes lipid trafficking at the ER-mitochondria contact sites (EMCS) through a non-conventional, unfolded protein response-independent, mechanism. PERK operates as an adaptor for the recruitment of the ER-plasma membrane tether and lipid transfer protein (LTP) Extended-Synaptotagmin 1 (E-Syt1), within the EMCS. In resting cells, the heterotypic E-Syt1-PERK interaction endorses transfer of PLs between the ER and mitochondria. Weakening the E-Syt1-PERK interaction or removing the lipid transfer SMP-domain of E-Syt1, compromises mitochondrial respiration. Our findings unravel E-Syt1 as a PERK interacting LTP and molecular component of the lipid trafficking machinery of the EMCS, which critically maintains mitochondrial homeostasis and fitness.
The tight cross talk between two essential organelles of the cell, the endoplasmic reticulum (ER) and mitochondria, is spatially and functionally regulated by specific microdomains known as the ...mitochondria-associated membranes (MAMs). MAMs are hot spots of Ca
transfer between the ER and mitochondria, and emerging data indicate their vital role in the regulation of fundamental physiological processes, chief among them mitochondria bioenergetics, proteostasis, cell death, and autophagy. Moreover, and perhaps not surprisingly, it has become clear that signaling events regulated at the ER-mitochondria intersection regulate key processes in oncogenesis and in the response of cancer cells to therapeutics. ER-mitochondria appositions have been shown to dynamically recruit oncogenes and tumor suppressors, modulating their activity and protein complex formation, adapt the bioenergetic demand of cancer cells and to regulate cell death pathways and redox signaling in cancer cells. In this review, we discuss some emerging players of the ER-mitochondria contact sites in mammalian cells, the key processes they regulate and recent evidence highlighting the role of MAMs in shaping cell-autonomous and non-autonomous signals that regulate cancer growth.
The endoplasmic reticulum (ER) is the main hub of cellular Ca(2+)signalling and protein synthesis and folding. The ER moreover is the central player in the formation of contact sites with other ...organelles and structures, including mitochondria, plasma membrane (PM) and endosomes. The most studied of these, the ER-mitochondria contact sites, are crucial regulators of cellular Ca(2+)homoeostasis, metabolism and cell death signalling. Protein kinase RNA-like ER kinase (PERK), an ER stress kinase and crucial signalling protein in the unfolded protein response (UPR), was found to be able to orchestrate contact sites between the ER and mitochondria and to be indispensable for the pre-apoptotic trafficking of calreticulin (CRT) at the PM during immunogenic cell death (ICD). Furthermore, PERK has recently been linked with ER and PM contact sites through the mechanism of store-operated Ca(2+)entry (SOCE). Here we discuss emerging findings disclosing novel roles of the ER stress sensor PERK in orchestrating inter-organellar communication in the context of ER stress.
Both human embryonic stem cells and induced pluripotent stem cells can self-renew indefinitely in culture; however, present methods to clonally grow them are inefficient and poorly defined for ...genetic manipulation and therapeutic purposes. Here we develop the first chemically defined, xeno-free, feeder-free synthetic substrates to support robust self-renewal of fully dissociated human embryonic stem and induced pluripotent stem cells. Material properties including wettability, surface topography, surface chemistry and indentation elastic modulus of all polymeric substrates were quantified using high-throughput methods to develop structure-function relationships between material properties and biological performance. These analyses show that optimal human embryonic stem cell substrates are generated from monomers with high acrylate content, have a moderate wettability and employ integrin alpha(v)beta(3) and alpha(v)beta(5) engagement with adsorbed vitronectin to promote colony formation. The structure-function methodology employed herein provides a general framework for the combinatorial development of synthetic substrates for stem cell culture.
Loss of ER Ca2+ homeostasis triggers endoplasmic reticulum (ER) stress and drives ER-PM contact sites formation in order to refill ER-luminal Ca2+. Recent studies suggest that the ER stress sensor ...and mediator of the unfolded protein response (UPR) PERK regulates intracellular Ca2+ fluxes, but the mechanisms remain elusive. Here, using proximity-dependent biotin identification (BioID), we identified the actin-binding protein Filamin A (FLNA) as a key PERK interactor. Cells lacking PERK accumulate F-actin at the cell edges and display reduced ER-PM contacts. Following ER-Ca2+ store depletion, the PERK-FLNA interaction drives the expansion of ER-PM juxtapositions by regulating F-actin-assisted relocation of the ER-associated tethering proteins Stromal Interaction Molecule 1 (STIM1) and Extended Synaptotagmin-1 (E-Syt1) to the PM. Cytosolic Ca2+ elevation elicits rapid and UPR-independent PERK dimerization, which enforces PERK-FLNA-mediated ER-PM juxtapositions. Collectively, our data unravel an unprecedented role of PERK in the regulation of ER-PM appositions through the modulation of the actin cytoskeleton.
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•The ER stress sensor PERK interacts with the actin regulator FLNA•Loss of PERK leads to a disturbed actin cytoskeleton and increased cortical F-actin•A functional PERK-FLNA axis is required for efficient ER-PM contact site formation•PERK is activated through cytosolic Ca2+ increase independent of the UPR
van Vliet et al. show that the actin regulator FLNA interacts with the ER stress kinase PERK and that this interaction is required for the efficient formation of ER-plasma membrane contact sites. This function of PERK is independent of the UPR and is activated by ER Ca2+ store depletion.
ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow ...between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.
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•ATG9A and ATG2A form a heterotetrameric complex•HDX and CXL-MS reveal intricate interaction interface between ATG9A and ATG2A•Disrupting the ATG9A-ATG2A complex disrupts autophagic flux•Lipid transfer tunnel of ATG2A binds proximal to the perpendicular branch of ATG9A
A critical aspect of autophagy is the membrane growth of the phagophore, a process that is still elusive. Here, we show that complex formation by the scramblase ATG9A and lipid transfer protein ATG2A is required for autophagy and characterize their interaction interface using structural mass spectrometry and EM techniques.
Excessive Ca2+ fluxes from the endoplasmic reticulum to the mitochondria result in apoptotic cell death. Bcl-2 and Bcl-XL proteins exert part of their anti-apoptotic function by directly targeting ...Ca2+-transport systems, like the endoplasmic reticulum-localized inositol 1,4,5-trisphosphate receptors (IP3Rs) and the voltage-dependent anion channel 1 (VDAC1) at the outer mitochondrial membranes. We previously demonstrated that the Bcl-2 homology 4 (BH4) domain of Bcl-2 protects against Ca2+-dependent apoptosis by binding and inhibiting IP3Rs, although the BH4 domain of Bcl-XL was protective independently of binding IP3Rs. Here, we report that in contrast to the BH4 domain of Bcl-2, the BH4 domain of Bcl-XL binds and inhibits VDAC1. In intact cells, delivery of the BH4-Bcl-XL peptide via electroporation limits agonist-induced mitochondrial Ca2+ uptake and protects against staurosporine-induced apoptosis, in line with the results obtained with VDAC1−/− cells. Moreover, the delivery of the N-terminal domain of VDAC1 as a synthetic peptide (VDAC1-NP) abolishes the ability of BH4-Bcl-XL to suppress mitochondrial Ca2+ uptake and to protect against apoptosis. Importantly, VDAC1-NP did not affect the ability of BH4-Bcl-2 to suppress agonist-induced Ca2+ release in the cytosol or to prevent apoptosis, as done instead by an IP3R-derived peptide. In conclusion, our data indicate that the BH4 domain of Bcl-XL, but not that of Bcl-2, selectively targets VDAC1 and inhibits apoptosis by decreasing VDAC1-mediated Ca2+ uptake into the mitochondria.
Background: VDAC1 mediates the transfer of pro-apoptotic Ca2+ signals into mitochondria.
Results: The BH4 domain of Bcl-XL, but not that of Bcl-2, targets VDAC1 and suppresses its pro-apoptotic Ca2+-flux properties. N-terminal VDAC1 peptide alleviates this effect of BH4-Bcl-XL.
Conclusion: Bcl-XL via its BH4 domain inhibits VDAC1 activity.
Significance: Bcl-2 and Bcl-XL differ in their BH4 domain biology by regulating ER and mitochondrial Ca2+-transport systems, respectively.
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