The Hob proteins are novel and conserved lipid binding proteins at ER-PM contact sites

ABSTRACTMembrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit/Hob proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in higher animals, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

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The Hob proteins are novel and conserved lipid-binding proteins at ER–PM contact sites

Journal of Cell Science ◽

10.1242/jcs.259086 ◽

2021 ◽

Vol 135 (5) ◽

Author(s):

Sarah D. Neuman ◽

Jeff R. Jorgensen ◽

Amy T. Cavanagh ◽

Jeremy T. Smyth ◽

Jane E. Selegue ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Composition ◽

Lipid Binding ◽

Model Systems ◽

Yeast Cells ◽

Membrane Contact Sites ◽

Contact Sites ◽

Critical Junctures ◽

C Terminus ◽

Membrane Contact

ABSTRACT Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum–plasma membrane (ER–PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER–PM contact sites. We find that Hobbit localizes to ER–PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

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Kv2 potassium channels form endoplasmic reticulum/plasma membrane junctions via interaction with VAPA and VAPB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1805757115 ◽

2018 ◽

Vol 115 (31) ◽

pp. E7331-E7340 ◽

Cited By ~ 46

Author(s):

Ben Johnson ◽

Ashley N. Leek ◽

Laura Solé ◽

Emily E. Maverick ◽

Tim P. Levine ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Neuronal Cultures ◽

Physical Relationship ◽

Membrane Contact Sites ◽

Contact Sites ◽

Hek Cells ◽

C Terminus ◽

Membrane Contact

Kv2.1 exhibits two distinct forms of localization patterns on the neuronal plasma membrane: One population is freely diffusive and regulates electrical activity via voltage-dependent K+ conductance while a second one localizes to micrometer-sized clusters that contain densely packed, but nonconducting, channels. We have previously established that these clusters represent endoplasmic reticulum/plasma membrane (ER/PM) junctions that function as membrane trafficking hubs and that Kv2.1 plays a structural role in forming these membrane contact sites in both primary neuronal cultures and transfected HEK cells. Clustering and the formation of ER/PM contacts are regulated by phosphorylation within the channel C terminus, offering cells fast, dynamic control over the physical relationship between the cortical ER and PM. The present study addresses the mechanisms by which Kv2.1 and the related Kv2.2 channel interact with the ER membrane. Using proximity-based biotinylation techniques in transfected HEK cells we identified ER VAMP-associated proteins (VAPs) as potential Kv2.1 interactors. Confirmation that Kv2.1 and -2.2 bind VAPA and VAPB employed colocalization/redistribution, siRNA knockdown, and Förster resonance energy transfer (FRET)-based assays. CD4 chimeras containing sequence from the Kv2.1 C terminus were used to identify a noncanonical VAP-binding motif. VAPs were first identified as proteins required for neurotransmitter release in Aplysia and are now known to be abundant scaffolding proteins involved in membrane contact site formation throughout the ER. The VAP interactome includes AKAPs, kinases, membrane trafficking machinery, and proteins regulating nonvesicular lipid transport from the ER to the PM. Therefore, the Kv2-induced VAP concentration at ER/PM contact sites is predicted to have wide-ranging effects on neuronal cell biology.

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Peroxisome retention involves Inp1-dependent peroxisome–plasma membrane contact sites in yeast

The Journal of Cell Biology ◽

10.1083/jcb.201906023 ◽

2020 ◽

Vol 219 (10) ◽

Author(s):

Arjen M. Krikken ◽

Huala Wu ◽

Rinse de Boer ◽

Damien P. Devos ◽

Tim P. Levine ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Cytoskeleton ◽

Hansenula Polymorpha ◽

Peroxisomal Membrane ◽

Membrane Contact Sites ◽

Contact Sites ◽

C Terminus ◽

Latrunculin A ◽

Membrane Contact ◽

Mother Cells

Retention of peroxisomes in yeast mother cells requires Inp1, which is recruited to the organelle by the peroxisomal membrane protein Pex3. Here we show that Hansenula polymorpha Inp1 associates peroxisomes to the plasma membrane. Peroxisome–plasma membrane contact sites disappear upon deletion of INP1 but increase upon INP1 overexpression. Analysis of truncated Inp1 variants showed that the C terminus is important for association to the peroxisome, while a stretch of conserved positive charges and a central pleckstrin homology-like domain are important for plasma membrane binding. In cells of a PEX3 deletion, strain Inp1-GFP localizes to the plasma membrane, concentrated in patches near the bud neck and in the cortex of nascent buds. Upon disruption of the actin cytoskeleton by treatment of the cells with latrunculin A, Inp1-GFP became cytosolic, indicating that Inp1 localization is dependent on the presence of an intact actin cytoskeleton.

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Faculty Opinions recommendation of Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.8941961.9505055 ◽

2011 ◽

Author(s):

Davis Ng ◽

Guillaume Thibault

Keyword(s):

Plasma Membrane ◽

Phosphoinositide Metabolism ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

Author(s):

Noemi Ruiz-Lopez ◽

Jessica Pérez-Sancho ◽

Alicia Esteban del Valle ◽

Richard P Haslam ◽

Steffen Vanneste ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Abiotic Stress ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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Sigma 1 Receptor, Cholesterol and Endoplasmic Reticulum Contact Sites

Contact ◽

10.1177/25152564211026505 ◽

2021 ◽

Vol 4 ◽

pp. 251525642110265

Author(s):

Vladimir Zhemkov ◽

Jen Liou ◽

Ilya Bezprozvanny

Keyword(s):

Endoplasmic Reticulum ◽

Protein Composition ◽

Sigma 1 Receptor ◽

Functional Roles ◽

Membrane Contact Sites ◽

Contact Sites ◽

Sigma 1 ◽

Membrane Contact ◽

Organelle Communication ◽

Cellular Organelles

Recent studies indicated potential importance of membrane contact sites (MCS) between the endoplasmic reticulum (ER) and other cellular organelles. These MCS have unique protein and lipid composition and serve as hubs for inter-organelle communication and signaling. Despite extensive investigation of MCS protein composition and functional roles, little is known about the process of MCS formation. In this perspective, we propose a hypothesis that MCS are formed not as a result of random interactions between membranes of ER and other organelles but on the basis of pre-existing cholesterol-enriched ER microdomains.

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Mdm1/Snx13 is a novel ER–endolysosomal interorganelle tethering protein

The Journal of Cell Biology ◽

10.1083/jcb.201503088 ◽

2015 ◽

Vol 210 (4) ◽

pp. 541-551 ◽

Cited By ~ 72

Author(s):

W. Mike Henne ◽

Lu Zhu ◽

Zsolt Balogi ◽

Christopher Stefan ◽

Jeffrey A. Pleiss ◽

...

Keyword(s):

Neurological Disease ◽

Lipid Binding ◽

Sphingolipid Metabolism ◽

Membrane Contact Sites ◽

Contact Sites ◽

Effector Genes ◽

Px Domain ◽

In Trans ◽

Membrane Contact ◽

Lysosome Membrane

Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain–containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)–vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER–vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER–vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease–associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.

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The Hob Proteins: Putative, Novel Lipid Transfer Proteins at ER-PM Contact Sites

Contact ◽

10.1177/25152564211052376 ◽

2021 ◽

Vol 4 ◽

pp. 251525642110523

Author(s):

Sarah D. Neuman ◽

Amy T. Cavanagh ◽

Arash Bashirullah

Keyword(s):

Structural Models ◽

Model Systems ◽

Lipid Transfer ◽

Lipid Transfer Proteins ◽

Membrane Contact Sites ◽

Contact Sites ◽

Bona Fide ◽

Membrane Contact ◽

Mutant Cells ◽

Critical Process

Nonvesicular transfer of lipids at membrane contact sites (MCS) has recently emerged as a critical process for cellular function. Lipid transfer proteins (LTPs) mediate this unique transport mechanism, and although several LTPs are known, the cellular complement of these proteins continues to expand. Our recent work has revealed the highly conserved but poorly characterized Hobbit/Hob proteins as novel, putative LTPs at endoplasmic reticulum-plasma membrane (ER-PM) contact sites. Using both S. cerevisiae and D. melanogaster model systems, we demonstrated that the Hob proteins localize to ER-PM contact sites via an N-terminal ER membrane anchor and conserved C-terminal sequences. These conserved C-terminal sequences bind to phosphoinositides (PIPs), and the distribution of PIPs is disrupted in hobbit mutant cells. Recently released structural models of the Hob proteins exhibit remarkable similarity to other bona fide LTPs, like VPS13A and ATG2, that function at MCS. Hobbit is required for viability in Drosophila, suggesting that the Hob proteins are essential genes that may mediate lipid transfer at MCS.

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