Peroxisomal Membrane Contact Sites in Mammalian Cells

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins, ACBD5 and ACBD4, and the ER protein vesicle-associated membrane protein-associated protein-B (VAPB) as tethering components, which physically interact to foster PO–ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of PO–ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into PO–ER associations at the level of membrane contact sites, their role, composition, and regulation, we have developed two fluorescence-based systems to monitor PO–ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay, we were able to measure the changes in PO–ER interactions while the split-fluorescence reporter was more limited and only allowed us to label PO–ER contacts. We show that both techniques can be useful additions to the toolkit of methods to study PO–ER associations and explore the relative merits of each.

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Regulating peroxisome–ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

The Journal of Cell Biology ◽

10.1083/jcb.202003143 ◽

2022 ◽

Vol 221 (3) ◽

Author(s):

Suzan Kors ◽

Christian Hacker ◽

Chloe Bolton ◽

Renate Maier ◽

Lena Reimann ◽

...

Keyword(s):

Membrane Protein ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Glycogen Synthase ◽

Current Model ◽

Protein Domain ◽

Major Sperm Protein ◽

Peroxisomal Membrane ◽

Membrane Contact Sites ◽

Contact Sites

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A–binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein–associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome–ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB—and thus peroxisome–ER contact sites—differently. Moreover, we demonstrate that GSK3β (glycogen synthase kinase-3 β) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome–ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.

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Pex3-mediated peroxisomal membrane contact sites in yeast

10.33612/diss.113450193 ◽

2020 ◽

Cited By ~ 1

Author(s):

Huala Wu

Keyword(s):

Peroxisomal Membrane ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

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ORP5 Transfers Phosphatidylserine To Mitochondria And Regulates Mitochondrial Calcium Uptake At Endoplasmic Reticulum - Mitochondria Contact Sites

10.1101/695577 ◽

2019 ◽

Cited By ~ 1

Author(s):

Leila Rochin ◽

Cécile Sauvanet ◽

Eeva Jääskeläinen ◽

Audrey Houcine ◽

Amita Arora ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Calcium Uptake ◽

Vesicular Transport ◽

Mitochondrial Calcium ◽

Mitochondrial Membranes ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Mitochondrial Calcium Uptake

SUMMARYMitochondria are dynamic organelles essential for cell survival whose structural and functional integrity rely on selective and regulated transport of lipids from/to the endoplasmic reticulum (ER) and across the two mitochondrial membranes. As they are not connected by vesicular transport, the exchange of lipids between ER and mitochondria occurs at sites of close organelle apposition called membrane contact sites. However, the mechanisms and proteins involved in these processes are only beginning to emerge. Here, we show that ORP5/8 mediate non-vesicular transport of Phosphatidylserine (PS) from the ER to mitochondria in mammalian cells. We also show that ER-mitochondria contacts where ORP5/8 reside are physically and functionally linked to the MIB/MICOS complexes that bridge the mitochondrial membranes, cooperating with them to facilitate PS transfer from the ER to the mitochondria. Finally, we show that ORP5 but not ORP8, additionally regulates import of calcium to mitochondria and consequently cell senescence.

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Peroxisomal Membrane Contact Sites in Yeasts

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.735031 ◽

2021 ◽

Vol 9 ◽

Author(s):

Amit S. Joshi

Keyword(s):

Lipid Droplets ◽

Peroxisome Biogenesis ◽

Functional Importance ◽

Peroxisomal Membrane ◽

Membrane Contact Sites ◽

Contact Sites ◽

Recent Developments ◽

Membrane Contacts ◽

Membrane Bound ◽

Membrane Contact

Peroxisomes are ubiquitous, single membrane-bound organelles that play a crucial role in lipid metabolism and human health. While peroxisome number is maintained by the division of existing peroxisomes, nascent peroxisomes can be generated from the endoplasmic reticulum (ER) membrane in yeasts. During formation and proliferation, peroxisomes maintain membrane contacts with the ER. In addition to the ER, contacts between peroxisomes and other organelles such as lipid droplets, mitochondria, vacuole, and plasma membrane have been reported. These membrane contact sites (MCS) are dynamic and important for cellular function. This review focuses on the recent developments in peroxisome biogenesis and the functional importance of peroxisomal MCS in yeasts.

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Di-arginine and FFAT-like motifs retain a subpopulation of PRA1 at ER-mitochondria membrane contact sites

10.1101/2020.01.27.922260 ◽

2020 ◽

Author(s):

Ameair Abu Irqeba ◽

Judith Mosinger Ogilvie

Keyword(s):

Mammalian Cells ◽

Protein Localization ◽

Transmembrane Protein ◽

Amino Acid Sequences ◽

Terminal Region ◽

Rab Gtpases ◽

Membrane Contact Sites ◽

Contact Sites ◽

Er Retention ◽

Membrane Contact

ABSTRACTPrenylated Rab Acceptor 1 (PRA1/Rabac1) is a four-pass transmembrane protein that has been found to localize to the Golgi and promiscuously associate with a diverse array of Rab GTPases. We have previously identified PRA1 to be among the earliest significantly down-regulated genes in the rd1 mouse model of retinitis pigmentosa, a retinal degenerative disease. Here, we show that an endogenous subpopulation of PRA1 resides within the endoplasmic reticulum (ER) at ER-mitochondria membrane contact sites in cultured mammalian cells. We also demonstrate that PRA1 contains two previously unidentified ER retention/retrieval amino acid sequences on its cytosolic N-terminal region: a membrane distal di-arginine motif and a novel membrane proximal FFAT-like motif. Using a truncation construct that lacks complete Golgi targeting information, we show that mutation of either motif leads to an increase in cell surface localization, while mutation of both motifs exhibits an additive effect. We also present evidence that illustrates that N- or C- terminal addition of a tag to full-length PRA1 leads to differential localization to either the Golgi or reticular ER, phenotypes that do not completely mirror endogenous protein localization. The presence of multiple ER retention motifs on the PRA1 N-terminal region further suggests that it has a functional role within the ER.

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OSBP-Related Protein Family in Lipid Transport over Membrane Contact Sites

Lipid Insights ◽

10.4137/lpi.s31726 ◽

2015 ◽

Vol 8s1 ◽

pp. LPI.S31726 ◽

Cited By ~ 15

Author(s):

Vesa M. Olkkonen

Keyword(s):

Endoplasmic Reticulum ◽

Mammalian Cells ◽

Binding Protein ◽

High Capacity ◽

Retrograde Transport ◽

Protein Family ◽

Oxysterol Binding Protein ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

Increasing evidence suggests that oxysterol-binding protein-related proteins (ORPs) localize at membrane contact sites, which are high-capacity platforms for inter-organelle exchange of small molecules and information. ORPs can simultaneously associate with the two apposed membranes and transfer lipids across the interbilayer gap. Oxysterol-binding protein moves cholesterol from the endoplasmic reticulum to trans-Golgi, driven by the retrograde transport of phosphatidylinositol-4-phosphate (PI4P). Analogously, yeast Osh6p mediates the transport of phosphatidylserine from the endoplasmic reticulum to the plasma membrane in exchange for PI4P, and ORP5 and -8 are suggested to execute similar functions in mammalian cells. ORPs may share the capacity to bind PI4P within their ligand-binding domain, prompting the hypothesis that bidirectional transport of a phosphoinositide and another lipid may be a common theme among the protein family. This model, however, needs more experimental support and does not exclude a function of ORPs in lipid signaling.

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Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3β

10.1101/2021.11.11.467785 ◽

2021 ◽

Author(s):

Suzan Kors ◽

Christian Hacker ◽

Chloe Bolton ◽

Renate Maier ◽

Lena Reimann ◽

...

Keyword(s):

Membrane Protein ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Glycogen Synthase ◽

Current Model ◽

Protein Domain ◽

Major Sperm Protein ◽

Peroxisomal Membrane ◽

Membrane Contact Sites ◽

Contact Sites

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 [acyl-coenzyme A-binding domain protein 5] and the ER-resident protein VAPB [vesicle-associated membrane protein-associated protein B]. ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like [two phenylalanines (FF) in an acidic tract] motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB and thus, peroxisome-ER contact sites differently. Moreover, we demonstrate that GSK3β [glycogen synthase kinase-3 beta] regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.

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The Unfolded Protein Response and Membrane Contact Sites: Tethering as a Matter of Life and Death?

Contact ◽

10.1177/2515256418770512 ◽

2018 ◽

Vol 1 ◽

pp. 251525641877051 ◽

Cited By ~ 3

Author(s):

Alexander R. van Vliet ◽

Maria Livia Sassano ◽

Patrizia Agostinis

Keyword(s):

Unfolded Protein Response ◽

Cell Fate ◽

Mammalian Cells ◽

Cell Fate Decisions ◽

Unfolded Protein ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

The Unfolded Protein Response ◽

Protein Response

The endoplasmic reticulum (ER) is the most extensive organelle of the eukaryotic cell and constitutes the major site of protein and lipid synthesis and regulation of intracellular Ca2+ levels. To exert these functions properly, the ER network is shaped in structurally and functionally distinct domains that dynamically remodel in response to intrinsic and extrinsic cues. Moreover, the ER establishes a tight communication with virtually all organelles of the cell through specific subdomains called membrane contact sites. These contact sites allow preferential, nonvesicular channeling of key biological mediators including lipids and Ca2+ between organelles and are harnessed by the ER to interface with and coregulate a variety of organellar functions that are vital to maintain homeostasis. When ER homeostasis is lost, a condition that triggers the activation of an evolutionarily conserved pathway called the unfolded protein response (UPR), the ER undergoes rapid remodeling. These dynamic changes in ER morphology are functionally coupled to the modulation or formation of contact sites with key organelles, such as mitochondria and the plasma membrane, which critically regulate cell fate decisions of the ER-stressed cells. Certain components of the UPR have been shown to facilitate the formation of contact sites through various mechanisms including remodeling of the actin cytoskeleton. In this review, we discuss old and emerging evidence linking the UPR machinery to contact site formation in mammalian cells and discuss their important role in cellular homeostasis.

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Roles for ER:endosome membrane contact sites in ligand-stimulated intraluminal vesicle formation

Biochemical Society Transactions ◽

10.1042/bst20170432 ◽

2018 ◽

Vol 46 (5) ◽

pp. 1055-1062 ◽

Cited By ~ 3

Author(s):

Louise H. Wong ◽

Emily R. Eden ◽

Clare E. Futter

Keyword(s):

Membrane Proteins ◽

Mammalian Cells ◽

Tyrosine Phosphatase ◽

Endosomal Sorting ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contacts ◽

Epidermal Growth ◽

Membrane Contact ◽

Multivesicular Endosomes

Multivesicular endosomes/bodies (MVBs) sort membrane proteins between recycling and degradative pathways. Segregation of membrane proteins onto intraluminal vesicles (ILVs) of MVBs removes them from the recycling pathway and facilitates their degradation following fusion of MVBs with lysosomes. Sorting of many cargos onto ILVs depends on the ESCRT (Endosomal Sorting Complex Required for Transport) machinery, although ESCRT-independent mechanisms also exist. In mammalian cells, efficient sorting of ligand-stimulated epidermal growth factor receptors onto ILVs also depends on the tyrosine phosphatase, PTP1B, an ER-localised enzyme that interacts with endosomal targets at membrane contacts between MVBs and the ER. This review focuses on the potential roles played by ER:MVB membrane contact sites in regulating ESCRT-dependent ILV formation.

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