scholarly journals Seipin traps triacylglycerols to facilitate their nanoscale clustering in the endoplasmic reticulum membrane

PLoS Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. e3000998
Author(s):  
Xavier Prasanna ◽  
Veijo T. Salo ◽  
Shiqian Li ◽  
Katharina Ven ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplet ◽  
In Silico ◽  
Lipid Membrane ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Binding Interface ◽  
Biomolecular Simulations ◽  
Hydrophobic Helices ◽  
Tag Clustering

Seipin is a disk-like oligomeric endoplasmic reticulum (ER) protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While the S166D-seipin mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

scholarly journals Seipin traps triacylglycerols to facilitate their nanoscale clustering in the ER membrane

2020 ◽  
Author(s):  
Xavier Prasanna ◽  
Veijo T. Salo ◽  
Shiqian Li ◽  
Katharina Ven ◽  
Helena Vihinen ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
In Silico ◽  
Lipid Membrane ◽  
Wild Type ◽  
Binding Interface ◽  
Biomolecular Simulations ◽  
Hydrophobic Helices ◽  
Tag Clustering ◽  
Er Membrane ◽  
In Cells

AbstractSeipin is a disk-like oligomeric ER protein important for lipid droplet (LD) biogenesis and triacylglycerol (TAG) delivery to growing LDs. Here we show through biomolecular simulations bridged to experiments that seipin can trap TAGs in the ER bilayer via the luminal hydrophobic helices of the protomers delineating the inner opening of the seipin disk. This promotes the nanoscale sequestration of TAGs at a concentration that by itself is insufficient to induce TAG clustering in a lipid membrane. We identify Ser166 in the α3 helix as a favored TAG occupancy site and show that mutating it compromises the ability of seipin complexes to sequester TAG in silico and to promote TAG transfer to LDs in cells. While seipin-S166D mutant colocalizes poorly with promethin, the association of nascent wild-type seipin complexes with promethin is promoted by TAGs. Together, these results suggest that seipin traps TAGs via its luminal hydrophobic helices, serving as a catalyst for seeding the TAG cluster from dissolved monomers inside the seipin ring, thereby generating a favorable promethin binding interface.

scholarly journals Deletions into an NH2-terminal hydrophobic domain result in secretion of rotavirus VP7, a resident endoplasmic reticulum membrane glycoprotein.

1985 ◽  
Vol 101 (6) ◽  
pp. 2199-2209 ◽  
Author(s):  
M S Poruchynsky ◽  
C Tyndall ◽  
G W Both ◽  
F Sato ◽  
A R Bellamy ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Integral Membrane Protein ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Positive Signal ◽  
Complex Type ◽  
Immunofluorescent Localization ◽  
Hydrophobic Domain ◽  
Rotavirus A ◽  
Altered Protein

Rotavirus, a non-enveloped reovirus, buds into the rough endoplasmic reticulum and transiently acquires a membrane. The structural glycoprotein, VP7, a 38-kD integral membrane protein of the endoplasmic reticulum (ER), presumably transfers to virus in this process. The gene for VP7 potentially encodes a protein of 326 amino acids which has two tandem hydrophobic domains at the NH2-terminal, each preceded by an in-frame ATG codon. A series of deletion mutants constructed from a full-length cDNA clone of the Simian 11 rotavirus VP7 gene were expressed in COS 7 cells. Products from wild-type, and mutants which did not affect the second hydrophobic domain of VP7, were localized by immunofluorescence to elements of the ER only. However, deletions affecting the second hydrophobic domain (mutants 42-61, 43-61, 47-61) showed immunofluorescent localization of VP7 which coincided with that of wheat germ agglutinin, indicating transport to the Golgi apparatus. Immunoprecipitable wild-type protein, or an altered protein lacking the first hydrophobic sequence, remained intracellular and endo-beta-N-acetylglucosaminidase H sensitive. In contrast, products of mutants 42-61, 43-61, and 47-61 were transported from the ER, and secreted. Glycosylation of the secreted molecules was inhibited by tunicamycin, resistant to endo-beta-N-acetylglucosaminidase H digestion and therefore of the N-linked complex type. An unglycosylated version of VP7 was also secreted. We suggest that the second hydrophobic domain contributes to a positive signal for ER location and a membrane anchor function. Secretion of the mutant glycoprotein implies that transport can be constitutive with the destination being dictated by an overriding compartmentalization signal.

scholarly journals Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus

2007 ◽  
Vol 88 (8) ◽  
pp. 2204-2213 ◽  
Author(s):  
Steeve Boulant ◽  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Core Protein ◽  
Virus Production ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
The Core ◽  
Phenylalanine Residue

In infected cells, hepatitis C virus (HCV) core protein is targeted to lipid droplets, which serve as intracellular storage organelles. Using a tissue culture system to generate infectious HCV, we have shown that the coating of lipid droplets by the core protein occurs in a time-dependent manner and coincides with higher rates of virus production. At earlier times, the protein was located at punctate sites in close proximity to the edge of lipid droplets. Investigations by using Z-stack analysis have shown that many lipid droplets contained a single punctate site that could represent positions where core transfers from the endoplasmic reticulum membrane to droplets. The effects of lipid droplet association on virus production were studied by introducing mutations into the domain D2, the C-terminal region of the core protein necessary for droplet attachment. Alteration of a phenylalanine residue that was crucial for lipid droplet association generated an unstable form of the protein that could only be detected in the presence of a proteasome inhibitor. Moreover, converting two proline residues in D2 to alanines blocked coating of lipid droplets by core, although the protein was directed to punctate sites that were indistinguishable from those observed at early times for wild-type core protein. Neither of these virus mutants gave rise to virus progeny. By contrast, mutation at a cysteine residue positioned 2 aa upstream of the phenylalanine residue did not affect lipid droplet localization and produced wild-type levels of infectious progeny. Taken together, our findings indicate that lipid droplet association by core is connected to virus production.

scholarly journals Sec61p Serves Multiple Roles in Secretory Precursor Binding and Translocation into the Endoplasmic Reticulum Membrane

1998 ◽  
Vol 9 (12) ◽  
pp. 3455-3473 ◽  
Author(s):  
Marinus Pilon ◽  
Karin Römisch ◽  
Dong Quach ◽  
Randy Schekman
Keyword(s):  
Endoplasmic Reticulum ◽  
Receptor Site ◽  
Multiple Roles ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Evolutionarily Conserved ◽  
Cold Sensitive ◽  
A Subunit

The evolutionarily conserved Sec61 protein complex mediates the translocation of secretory proteins into the endoplasmic reticulum. To investigate the role of Sec61p, which is the main subunit of this complex, we generated recessive, cold-sensitive alleles ofsec61 that encode stably expressed proteins with strong defects in translocation. The stage at which posttranslational translocation was blocked was probed by chemical crosslinking of radiolabeled secretory precursors added to membranes isolated from wild-type and mutant strains. Two classes of sec61mutants were distinguished. The first class of mutants was defective in preprotein docking onto a receptor site of the translocon that included Sec61p itself. The second class of mutants allowed docking of precursors onto the translocon but was defective in the ATP-dependent release of precursors from this site that in wild-type membranes leads to pore insertion and full translocation. Only mutants of the second class were partially suppressed by overexpression ofSEC63, which encodes a subunit of the Sec61 holoenzyme complex responsible for positioning Kar2p (yeast BiP) at the translocation channel. These mutants thus define two early stages of translocation that require SEC61 function before precursor protein transfer across the endoplasmic reticulum membrane.

scholarly journals Dictyostelium Lipid Droplets Host Novel Proteins

2013 ◽  
Vol 12 (11) ◽  
pp. 1517-1529 ◽  
Author(s):  
Xiaoli Du ◽  
Caroline Barisch ◽  
Peggy Paschke ◽  
Cornelia Herrfurth ◽  
Oliver Bertinetti ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Unsaturated Fatty Acids ◽  
Neutral Lipids ◽  
Saturated Fatty Acids ◽  
Endoplasmic Reticulum Membrane ◽  
Hydrophobic Core ◽  
Content Type

ABSTRACT Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium , and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane.

scholarly journals Syntaxin 17 Recruits ACSL3 to Lipid Microdomains in Lipid Droplet Biogenesis

Contact ◽  
2019 ◽  
Vol 2 ◽  
pp. 251525641983871
Author(s):  
Hana Kimura ◽  
Kohei Arasaki ◽  
Moe Iitsuka ◽  
Mitsuo Tagaya
Keyword(s):  
Endoplasmic Reticulum ◽  
Lipid Droplet ◽  
Cholesterol Depletion ◽  
Endoplasmic Reticulum Membrane ◽  
Lipid Microdomains ◽  
Ceramide Synthesis ◽  
Detergent Resistant Membranes ◽  
A Minor ◽  
Ganglioside Gd3 ◽  
Neutral Lipid Biosynthesis

During lipid droplet (LD) formation, several key enzymes for neutral lipid biosynthesis, such as acyl-CoA synthetase 3 (ACSL3), translocate from the bilayer of the endoplasmic reticulum membrane or mitochondria-associated membrane to the monolayer surface of LDs. It has been recently shown that syntaxin 17 (Stx17) in cooperation with synaptosomal-associated protein of 23 kDa (SNAP23) facilitates the translocation of ACSL3 from the endoplasmic reticulum/mitochondria-associated membrane to LDs. In this study, we investigated whether lipid microdomains enriched in cholesterol and sphingolipids are important for the formation of LDs and the interaction of Stx17 with ACSL3 and SNAP23. Cholesterol depletion and blockage of ceramide synthesis by chemicals inhibited oleic acid (OA)-induced LD biogenesis and decreased the interaction of Stx17 with ACSL3 and SNAP23, whereas blockage of ganglioside GD3 synthesis by sialyltransferase knockdown interfered with LD biogenesis by affecting the interaction of Stx17 with SNAP23 but not ACSL3. Consistent with the requirement of GD3 in LD biogenesis, Stx17 was found to associate with GD3-containing membranes upon OA loading. SNAP23 and a minor fraction of Stx17 were found to reside in detergent-resistant membranes (DRMs), whereas OA treatment caused redistribution of ACSL3 and Stx17 to DRMs. Importantly, the redistribution of ACSL3 to DRMs was abrogated upon depletion of Stx17 or SNAP23. Taken together, our results highlight the importance of lipid microdomains enriched in cholesterol and sphingolipids as a platform for the interaction of Stx17 with ACSL3 and SNAP23 in LD biogenesis.

scholarly journals Gp78 E3 ubiquitin ligase mediates both basal and damage-induced mitophagy

2018 ◽  
Author(s):  
Bharat Joshi ◽  
Yayha Mohammadzadeh ◽  
Guang Gao ◽  
Ivan R. Nabi
Keyword(s):  
Endoplasmic Reticulum ◽  
Ubiquitin Ligase ◽  
Mitochondrial Fission ◽  
E3 Ubiquitin Ligase ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
Dual Roles ◽  
Mitochondrial Homeostasis ◽  
Fibrosarcoma Cells ◽  
Mitochondrial Volume

AbstractMitophagy, the elimination of mitochondria by the autophagy machinery, evolved to monitor mitochondrial health and maintain mitochondrial integrity. PINK1 is a sensor of mitochondrial health that recruits Parkin and other mitophagy-inducing ubiquitin ligases to depolarized mitochondria. However, mechanisms underlying mitophagic control of mitochondrial homeostasis, basal mitophagy, remain poorly understood. The Gp78 E3 ubiquitin ligase, an endoplasmic reticulum membrane protein, induces mitochondrial fission, endoplasmic reticulum-mitochondria contacts and mitophagy of depolarized mitochondria. CRISPR/Cas9 knockout of Gp78 in HT-1080 fibrosarcoma cells results in reduced ER-mitochondria contacts, increased mitochondrial volume and resistance to CCCP-induced mitophagy. Knockdown (KD) of the essential autophagy protein ATG5 increased mitochondrial volume of wild-type cells but did not impact mitochondrial volume of Gp78 knockout cells. This suggests that endogenous Gp78 actively eliminates mitochondria by autophagy in wild-type HT-1080 cells. Damage-induced mitophagy of depolarized mitochondria, in the presence of CCCP, but not basal mitophagy was prevented by knockdown of PINK1. This suggests that endogenous Gp78 plays dual roles in mitophagy induction: 1) control of mitochondrial homeostasis through mitophagy of undamaged mitochondria; and 2) elimination of damaged mitochondria through PINK1.

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