The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Lorenzo Frigerio - One of the best experts on this subject based on the ideXlab platform.
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bacterial expression purification and biophysical characterization of the smallest plant Reticulon isoform rtnlb13
Protein Expression and Purification, 2018Co-Authors: Michael Chow, Meropi Sklepari, Lorenzo Frigerio, Ann M DixonAbstract:Abstract Reticulons are a large family of integral membrane proteins that are ubiquitous in eukaryotes and play a key role in functional remodelling of the endoplasmic reticulum membrane. The Reticulon family is especially large in plants, with the Arabidopsis thaliana genome containing twenty-one isoforms. Reticulons vary in length but all contain a conserved C-terminal Reticulon homology domain (RHD) that associates with membranes. An understanding of the structure and membrane interactions of RHDs is key to unlocking their mechanism of function, however no three-dimensional structure has been solved. We believe that this is, in part, due to difficulties in obtaining Reticulon proteins in yields sufficient for structural study. To address this, we report here the first bacterial overexpression, purification, and biophysical investigation of a Reticulon protein from plants, the RTNLB13 protein from A. thaliana. RTNLB13 is the smallest plant Reticulon and is made up of a single RHD. We used circular dichroism, SDS-PAGE and analytical ultracentrifugation to reveal that RTNLB13 is 45% α-helical in a number of detergent environments, monomeric at low concentrations, and capable of self-association at higher concentrations. We used solution-state NMR to screen the effect of detergent type on the fold of isotopically-enriched RTNLB13, and found that ∼60% of the expected protein peaks were broadened due to slow dynamics. This broadening points toward a large network of protein-membrane interactions throughout the sequence. We have interpreted our results in light of current literature and suggest a preliminary description of RTNLB13 structure and topology.
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an arabidopsis Reticulon and the atlastin homologue rhd3 like2 act together in shaping the tubular endoplasmic reticulum
New Phytologist, 2013Co-Authors: Imogen Sparkes, Chris Hawes, Stefano Gattolin, Natasha Dzimitrowicz, Lynne M Roberts, Lorenzo FrigerioAbstract:Summary The endoplasmic reticulum (ER) is a network of membrane sheets and tubules connected via three-way junctions. A family of proteins, the Reticulons, are responsible for shaping the tubular ER. Reticulons interact with other tubule-forming proteins (Dp1 and Yop1p) and the GTPase atlastin. The Arabidopsis homologue of Dp1/Yop1p is HVA22. We show here that a seed-specific isoform of HVA22 labels the ER in tobacco (Nicotiana tabacum) cells but its overexpression does not alter ER morphology. The closest plant homologue of atlastin is RHD3. We show that RHD3-like 2 (RL2), the seed-specific isoform of RHD3, locates to the ER without affecting its shape or Golgi mobility. Expression of RL2-bearing mutations within its GTPase domain induces the formation of large ER strands, suggesting that a functional GTPase domain is important for the formation of three-way junctions. Coexpression of the Reticulon RTNLB13 with RL2 resulted in a dramatic alteration of the ER network. This alteration did not depend on an active GTPase domain but required a functional Reticulon, as no effect on ER morphology was seen when RL2 was coexpressed with a nonfunctional RTNLB13. RL2 and its GTPase mutants coimmunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 act together in modulating ER morphology.
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transmembrane domain length is responsible for the ability of a plant Reticulon to shape endoplasmic reticulum tubules in vivo
Plant Journal, 2010Co-Authors: Nicholas Tolley, Chris Hawes, Imogen Sparkes, Christian P Craddock, Peter J Eastmond, John Runions, Lorenzo FrigerioAbstract:Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape the ER into tubules. It has been hypothesized that their unusually long conserved hydrophobic regions cause Reticulons to assume a wedge-like topology that induces membrane curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana Reticulon (RTNLB13) localized to, and induced constrictions in, cortical ER tubules. Ectopic expression of RTNLB13 was sufficient to induce ER tubulation in an Arabidopsis mutant (pah1 pah2) whose ER membrane is mostly present in a sheet-like form. By sequential shortening of the four transmembrane domains (TMDs) of RTNLB13, we show that the length of the transmembrane regions is directly correlated with the ability of RTNLB13 to induce membrane tubulation and to form low-mobility complexes within the ER membrane. We also show that full-length TMDs are necessary for the ability of RTNLB13 to reside in the ER membrane.
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five arabidopsis Reticulon isoforms share endoplasmic reticulum location topology and membrane shaping properties
The Plant Cell, 2010Co-Authors: Imogen Sparkes, Stanley W Botchway, Nicholas Tolley, Isabel Aller, Julia Svozil, Anne Osterrieder, Christopher Mueller, Lorenzo FrigerioAbstract:The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana Reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Forster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete Reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.
Gia K Voeltz - One of the best experts on this subject based on the ideXlab platform.
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Reticulon short hairpin transmembrane domains are used to shape er tubules
Traffic, 2011Co-Authors: Nesia Zurek, Lenore Sparks, Gia K VoeltzAbstract:Reticulons are integral membrane proteins that partition into and shape the tubular endoplasmic reticulum (ER). We propose that Reticulons use a membrane insertion mechanism to generate regions of high membrane curvature in the ER. A Reticulon contains two short hairpin transmembrane domains (TMDs), which could generate membrane curvature by increasing the area of the cytoplasmic leaflet. Here, we test whether the short length of these hairpin TMDs is required for Reticulon membrane-shaping functions in mammalian cells. We lengthened the TMDs of Reticulon 4 to resemble a typical bi-pass TMD that spans both leaflets. We find that TMD mutants oligomerize like wild type (wt), however, they are not immobilized, do not partition into tubules, do not constrict tubules and no longer suppress peripheral ER cisternae. Therefore, short hairpin TMD length is required for Reticulon protein partitioning and membrane-shaping functions. Another membrane protein with a short hairpin TMD is caveolin. We show that an ER-retained caveolin construct also partitions within the ER in a manner that is dependent on it containing a short hairpin TMD. These data suggest that a short hairpin TMD may be a general feature used by membrane-shaping proteins to partition into and shape regions of high membrane curvature.
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the Reticulon and dp1 yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum
Journal of Biological Chemistry, 2008Co-Authors: Yoko Shibata, William A Prinz, Julia M Rist, Tom A Rapoport, Christiane Voss, Junjie Hu, Gia K VoeltzAbstract:We recently identified a class of membrane proteins, the Reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian Reticulons and DP1 are also relatively immobile and can form oligomers. The conserved Reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the Reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the Reticulons and DP1. We propose that oligomerization of the Reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules.
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The Reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum.
Journal of Biological Chemistry, 2008Co-Authors: Yoko Shibata, William A Prinz, Julia M Rist, Tom A Rapoport, Christiane Voss, Junjie Hu, Gia K VoeltzAbstract:We recently identified a class of membrane proteins, the Reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian Reticulons and DP1 are also relatively immobile and can form oligomers. The conserved Reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the Reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the Reticulons and DP1. We propose that oligomerization of the Reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules.
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a class of membrane proteins shaping the tubular endoplasmic reticulum
Cell, 2006Co-Authors: Gia K Voeltz, William A Prinz, Yoko Shibata, Julia M Rist, Tom A RapoportAbstract:SUMMARY How is the characteristic shape of a membrane bound organelle achieved? We have used an in vitro system to address the mechanism by which the tubular network of the endoplasmic reticulum (ER) is generated and maintained. Based on the inhibitory effect of sulfhydryl reagents and antibodies, network formation in vitro requires the integral membrane protein Rtn4a/ NogoA, a member of the ubiquitous Reticulon family. Both in yeast and mammalian cells, the Reticulons are largely restricted to the tubular ER and are excluded from the continuous sheets of the nuclear envelope and peripheral ER. Upon overexpression, the Reticulons form tubular membrane structures. The Reticulons interact with DP1/Yop1p, a conserved integral membraneproteinthatalsolocalizestothetubular ER. These proteins share an unusual hairpin topology in the membrane. The simultaneous absence of the Reticulons and Yop1p in S. cerevisiae results in disrupted tubular ER. We propose that these ‘‘morphogenic’’ proteins partition into and stabilize highly curved ER membrane tubules.
Stephen M. Strittmatter - One of the best experts on this subject based on the ideXlab platform.
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Reticulon 4a nogo a redistributes protein disulfide isomerase to protect mice from sod1 dependent amyotrophic lateral sclerosis
The Journal of Neuroscience, 2009Co-Authors: Yvonne S. Yang, Noam Y Harel, Stephen M. StrittmatterAbstract:Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease inherited in a small subset of patients. The SOD1(G93A) transgenic mouse models this subset of patients, and studies of this strain have suggested that endoplasmic reticulum (ER) stress and deficits in ER chaperone function are contributors to ALS pathophysiology. Here, we demonstrate that the Reticulon family of proteins is a novel regulator of the ER chaperone protein disulfide isomerase (PDI), and that through PDI, Reticulon-4A (Nogo-A) can protect mice against the neurodegeneration that characterizes ALS. We show that overexpressing Reticulon protein induces a punctate redistribution of PDI intracellularly, both in vitro and in vivo. Conversely, reduction of endogenous NogoA expression causes a more homogeneous expression pattern in vivo. These effects occur without induction of the unfolded protein response. To examine the effect of PDI redistribution on ALS disease progression, we conducted survival and behavior studies of SOD1(G93A) mice. Deletion of a single copy of the NogoA,B gene accelerates disease onset and progression, while deletion of both copies further worsens disease. We conclude that NogoA contributes to the proper function of the ER resident chaperone PDI, and is protective against ALS-like neurodegeneration. Our results provide a novel intracellular role for Reticulon proteins and support the hypothesis that modulation of PDI function is a potential therapeutic approach to ALS.
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The Reticulons: a family of proteins with diverse functions
Genome Biology, 2007Co-Authors: Yvonne S. Yang, Stephen M. StrittmatterAbstract:The Reticulon family is a large and diverse group of membrane-associated proteins found throughout the eukaryotic kingdom. All of its members contain a carboxy-terminal Reticulon homology domain that consists of two hydrophobic regions flanking a hydrophilic loop of 60-70 amino acids, but Reticulon amino-terminal domains display little or no similarity to each other. Reticulons principally localize to the endoplasmic reticulum, and there is evidence that they influence endoplasmic reticulum-Golgi trafficking, vesicle formation and membrane morphogenesis. However, mammalian Reticulons have also been found on the cell surface and mammalian Reticulon 4 expressed on the surface of oligodendrocytes is an inhibitor of axon growth both in culture and in vivo. There is also growing evidence that Reticulons may be important in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. The diversity of structure, topology, localization and expression patterns of Reticulons is reflected in their multiple, diverse functions in the cell.
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identification of the nogo inhibitor of axon regeneration as a Reticulon protein
Nature, 2000Co-Authors: Tadzia Grandpre, Fumio Nakamura, Timothy Vartanian, Stephen M. StrittmatterAbstract:Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts1. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth2. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described3,4,5,6,7. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury8,9. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.
Imogen Sparkes - One of the best experts on this subject based on the ideXlab platform.
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an arabidopsis Reticulon and the atlastin homologue rhd3 like2 act together in shaping the tubular endoplasmic reticulum
New Phytologist, 2013Co-Authors: Imogen Sparkes, Chris Hawes, Stefano Gattolin, Natasha Dzimitrowicz, Lynne M Roberts, Lorenzo FrigerioAbstract:Summary The endoplasmic reticulum (ER) is a network of membrane sheets and tubules connected via three-way junctions. A family of proteins, the Reticulons, are responsible for shaping the tubular ER. Reticulons interact with other tubule-forming proteins (Dp1 and Yop1p) and the GTPase atlastin. The Arabidopsis homologue of Dp1/Yop1p is HVA22. We show here that a seed-specific isoform of HVA22 labels the ER in tobacco (Nicotiana tabacum) cells but its overexpression does not alter ER morphology. The closest plant homologue of atlastin is RHD3. We show that RHD3-like 2 (RL2), the seed-specific isoform of RHD3, locates to the ER without affecting its shape or Golgi mobility. Expression of RL2-bearing mutations within its GTPase domain induces the formation of large ER strands, suggesting that a functional GTPase domain is important for the formation of three-way junctions. Coexpression of the Reticulon RTNLB13 with RL2 resulted in a dramatic alteration of the ER network. This alteration did not depend on an active GTPase domain but required a functional Reticulon, as no effect on ER morphology was seen when RL2 was coexpressed with a nonfunctional RTNLB13. RL2 and its GTPase mutants coimmunoprecipitate with RTNLB13. These results indicate that RL2 and RTNLB13 act together in modulating ER morphology.
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transmembrane domain length is responsible for the ability of a plant Reticulon to shape endoplasmic reticulum tubules in vivo
Plant Journal, 2010Co-Authors: Nicholas Tolley, Chris Hawes, Imogen Sparkes, Christian P Craddock, Peter J Eastmond, John Runions, Lorenzo FrigerioAbstract:Reticulons are integral endoplasmic reticulum (ER) membrane proteins that have the ability to shape the ER into tubules. It has been hypothesized that their unusually long conserved hydrophobic regions cause Reticulons to assume a wedge-like topology that induces membrane curvature. Here we provide proof of this hypothesis. When over-expressed, an Arabidopsis thaliana Reticulon (RTNLB13) localized to, and induced constrictions in, cortical ER tubules. Ectopic expression of RTNLB13 was sufficient to induce ER tubulation in an Arabidopsis mutant (pah1 pah2) whose ER membrane is mostly present in a sheet-like form. By sequential shortening of the four transmembrane domains (TMDs) of RTNLB13, we show that the length of the transmembrane regions is directly correlated with the ability of RTNLB13 to induce membrane tubulation and to form low-mobility complexes within the ER membrane. We also show that full-length TMDs are necessary for the ability of RTNLB13 to reside in the ER membrane.
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five arabidopsis Reticulon isoforms share endoplasmic reticulum location topology and membrane shaping properties
The Plant Cell, 2010Co-Authors: Imogen Sparkes, Stanley W Botchway, Nicholas Tolley, Isabel Aller, Julia Svozil, Anne Osterrieder, Christopher Mueller, Lorenzo FrigerioAbstract:The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana Reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Forster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete Reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.
Stanley W Botchway - One of the best experts on this subject based on the ideXlab platform.
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the odd one out arabidopsis Reticulon 20 does not bend er membranes but has a role in lipid regulation
Scientific Reports, 2018Co-Authors: Verena Kriechbaumer, Lilly Manetapeyret, Stanley W Botchway, Jessica Upson, Louise Hughes, Jake Richardson, Maike Kittelmann, Laetitia Fouillen, Patrick MoreauAbstract:Reticulons are integral ER membrane proteins characterised by a Reticulon homology domain comprising four transmembrane domains which results in the proteins sitting in the membrane in a W-topology. Here we report on a novel subgroup of Reticulons with an extended N-terminal domain and in particular on arabidopsis Reticulon 20. Using high resolution confocal microscopy we show that Reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue Reticulon 19 labels the whole ER. Other than demonstrated for the other members of the Reticulon protein family RTN20 and 19 do not display ER constriction phenotypes on over expression. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in roots indicating a role in lipid regulation. A third homologue in this family -3BETAHSD/D1- is unexpectedly localised to ER exit sites resulting in an intriguing location difference for the three proteins.
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the odd one out arabidopsis Reticulon 20 has a role in lipid biosynthesis
bioRxiv, 2017Co-Authors: Verena Kriechbaumer, Lilly Manetapeyret, Stanley W Botchway, Jessica Upson, Louise Hughes, Jake Richardson, Maike Kittelmann, Patrick Moreau, Chris HawesAbstract:The family of Reticulon proteins has been shown to be involved in a variety of functions in eukaryotic cells including tubulation of the endoplasmic reticulum (ER), formation of cell plates and primary plasmodesmata. Reticulons are integral ER membrane proteins characterised by a Reticulon homology domain comprising four transmembrane domains which results in the Reticulons sitting in the membrane in a W-topology. Here we report on a subgroup of Reticulons with an extended N-terminal domain and in particular on Arabidopsis Reticulon 20. We show that Reticulon 20 is located in a unique punctate pattern on the ER membrane. Its closest homologue Reticulon 19 labels the whole ER. We show that mutants in RTN20 or RTN19, respectively, display a significant change in sterol composition in the roots indicating a role in lipid biosynthesis or regulation. A third homologue in this family — 3BETAHSD/D1 — is localised to ER exit sites resulting in an intriguing location difference for the three proteins.
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a c terminal amphipathic helix is necessary for the in vivo tubule shaping function of a plant Reticulon
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Emily Breeze, Verena Kriechbaumer, Stanley W Botchway, Chris Hawes, Ann M Dixon, Natasha Dzimitrowicz, Rhiannon L Brooks, Jacob P Brady, Jason R Schnell, Mark D FrickerAbstract:Reticulons (RTNs) are a class of endoplasmic reticulum (ER) membrane proteins that are capable of maintaining high membrane curvature, thus helping shape the ER membrane into tubules. The mechanism of action of RTNs is hypothesized to be a combination of wedging, resulting from the transmembrane topology of their conserved Reticulon homology domain, and scaffolding, arising from the ability of RTNs to form low-mobility homo-oligomers within the membrane. We studied the plant RTN isoform RTN13, which has previously been shown to locate to ER tubules and the edges of ER cisternae and to induce constrictions in ER tubules when overexpressed, and identified a region in the C terminus containing a putative amphipathic helix (APH). Here we show that deletion of this region or disruption of the hydrophobic face of the predicted helix abolishes the ability of RTN13 to induce constrictions of ER tubules in vivo. These mutants, however, still retain their ability to interact and form low-mobility oligomers in the ER membrane. Hence, our evidence indicates that the conserved APH is a key structural feature for RTN13 function in vivo, and we propose that RTN, like other membrane morphogens, rely on APHs for their function.
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five arabidopsis Reticulon isoforms share endoplasmic reticulum location topology and membrane shaping properties
The Plant Cell, 2010Co-Authors: Imogen Sparkes, Stanley W Botchway, Nicholas Tolley, Isabel Aller, Julia Svozil, Anne Osterrieder, Christopher Mueller, Lorenzo FrigerioAbstract:The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana Reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Forster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete Reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.