Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

Ostermeyer, Anne G.; Ramcharan, Lynne T.; Zeng, Yifeng; Lublin, Douglas M.; Brown, Deborah A.

doi:10.1083/jcb.200303037

Cited by 69 publications

(86 citation statements)

References 52 publications

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Section: Discussionmentioning

confidence: 99%

“…Although caveolins differ from viperin in that they encode two hydrophobic domains that insert into membranes and generate a cytosolic hairpin, altering hydrophobic residues within these domains prevented caveolin-1 or caveolin-2 from associating with lipid droplets (18,19). Furthermore, these analyses indicated that the packing of the hydrophobic residues in these domains is specifically important for localizing to lipid droplets, as altering some of these residues did not affect association with other membranes, such as the plasma membrane and Golgi (19). Because lipid droplets are derived from ER membranes, the packing and hydrophobicity of the N-terminal amphipathic ␣-helices of viperin and NS5A may be suited for binding to both the ER and lipid droplets and may facilitate their localization to lipid droplets in the context of infection.…”

Section: Discussionmentioning

confidence: 99%

“…Some of the best characterized are the hydrophobic domains of caveolin-1 and caveolin-2, which have been shown to be necessary to target these proteins to lipid droplets (18,19). Although caveolins differ from viperin in that they encode two hydrophobic domains that insert into membranes and generate a cytosolic hairpin, altering hydrophobic residues within these domains prevented caveolin-1 or caveolin-2 from associating with lipid droplets (18,19). Furthermore, these analyses indicated that the packing of the hydrophobic residues in these domains is specifically important for localizing to lipid droplets, as altering some of these residues did not affect association with other membranes, such as the plasma membrane and Golgi (19).…”

Section: Discussionmentioning

confidence: 99%

See 2 more Smart Citations

The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

Hinson¹,

Cresswell

2009

Proc. Natl. Acad. Sci. U.S.A.

208

197

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Lipid droplets are intracellular lipid-storage organelles that are thought to be derived from the endoplasmic reticulum (ER). Several pathogens, notably hepatitis C virus, use lipid droplets for replication. Numerous questions remain about how lipid droplets are generated and used by viruses. Here we show that the IFN-induced antiviral protein viperin, which localizes to the cytosolic face of the ER and inhibits HCV, localizes to lipid droplets. We show that the N-terminal amphipathic ␣-helix of viperin that is responsible for ER localization is also necessary and sufficient to localize both viperin and the fluorescent protein dsRed to lipid droplets. Point mutations in the ␣-helix that prevent ER association also disrupt lipid droplet association, and sequential deletion mutants indicate that the same number of helical turns are necessary for ER and lipid droplet association. Finally, we show that the N-terminal amphipathic ␣-helix of the hepatitis C viral protein NS5A can localize dsRed and viperin to lipid droplets. These findings indicate that the amphipathic ␣-helices of viperin and NS5A are lipid droplet-targeting domains and suggest that viperin inhibits HCV by localizing to lipid droplets using a domain and mechanism similar to that used by HCV itself.HCV ͉ NS5A ͉ L ipid droplets consist of a core of neutral lipids surrounded by an outer phospholipid monolayer and associated proteins. These organelles are thought to be generated when neutral lipids accumulate in the endoplasmic reticulum (ER) bilayer. The mass of lipids is believed to bulge from the ER membrane into the cytoplasm, with the cytosolic leaflet of the ER membrane forming the outer phospholipid layer of the lipid droplet (1, 2). Recently, lipid droplets have been shown to play a role in several cellular processes, including lipid storage, lipid trafficking, and protein storage and degradation. The importance of this organelle is underscored by the fact that lipid droplets have been linked to several metabolic diseases, most notably diabetes and obesity (1).Lipid droplets have been shown to play a critical role in the replication of several pathogens. One of the most wellcharacterized examples is hepatitis C virus (HCV) (3, 4). Upon infection, the HCV core initially localizes to cytosolic face of the ER. After maturation, which requires two cleavage events, the core localizes to lipid droplets by a newly exposed domain, D2, which contains two amphipathic ␣-helices (5, 6). The core recruits the HCV nonstructural (NS) proteins and HCV replication complexes to lipid droplets (4). Several of these NS proteins localize to the ER before lipid droplet recruitment and contain amphipathic ␣-helices that are thought to facilitate interactions with the ER (7-9). In addition, HCV is thought to bring ER membranes in close proximity to the lipid droplet to create a localized environment with a high concentration of membranes for viral replication and assembly (4).Viperin is an IFN-induced antiviral protein that is induced upon HCV infection and inhibits HCV...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

Hinson¹,

Cresswell

2009

Proc. Natl. Acad. Sci. U.S.A.

208

197

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“…DGAT2 has a potential proline knot with 5 proline residues from amino acids 329 and 350. Other proteins, including perilipin, caveolin, AAM-B, and viperin are directed to lipid droplets by distinct hydrophobic sequences (33,(63)(64)(65). DGAT2 has a moderately hydrophobic region between amino acids 230 and 250 that may interact with the lipid droplet surface (20).…”

Section: Discussionmentioning

confidence: 99%

Murine Diacylglycerol Acyltransferase-2 (DGAT2) Can Catalyze Triacylglycerol Synthesis and Promote Lipid Droplet Formation Independent of Its Localization to the Endoplasmic Reticulum

McFie

Banman

Kary

et al. 2011

Journal of Biological Chemistry

132

114

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Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acylCoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER. Triacylglycerols (TG),2 the major form of stored energy in eukaryotic organisms, are synthesized via the multistep glycerol 3-phosphate or Kennedy pathway (1). In this pathway, three fatty acyl-coenzyme A (CoA) molecules (activated forms of fatty acids) are linked via ester bonds to a glycerol backbone. The microsomal enzyme, acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the final reaction of the pathway by forming an ester bond between a long chain fatty acid and the free hydroxyl group of diacylglycerol (DG) generating TG.Two different DGAT enzymes, DGAT1 and DGAT2, are responsible for the acyl-CoA-dependent synthesis of TG in mammalian tissues (2-4). The genes encoding these two enzymes have no sequence homology and they each belong to distinct gene families. DGAT1 belongs to a large family of membrane-bound O-acyltransferases that includes acyl-CoA: cholesterol acyltransferase (ACAT)-1 and -2, which catalyze cholesterol ester biosynthesis (5-9). DGAT2 belongs to the DGAT2/acyl-CoA:monoacylglycerol acyltransferase gene family that includes monoacylglycerol acyl-CoA acyltransferases 1-3 and wax synthase (3, 8, 10 -14). In mice and humans, DGAT1 and DGAT2 are expressed in most tissues, with the highest levels of expression found in tissues that are associated with TG metabolism (adipose tissue, liver, small intestine, and mammary gland) (3, 5).Studies in mice and in cells in culture have provided compelling evidence that DGAT2, more so than DGAT1, is responsible for the majority of TG synthesis and that these two enzymes have separa...

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“…5B) even though the N terminus of caveolin-1 is supposed to be exposed for digestion. Caveolin-1 adopts a hairpin topology with both ends facing the cytosolic side of the plasma membrane (21,22). If the membranes were ruptured by 0.5% CHAPS, caveolin-1 and the RTN3 fragments would be further digested by proteinase K (Fig.…”

Section: The Orientation Of the C-terminalmentioning

confidence: 99%

The Membrane Topology of RTN3 and Its Effect on Binding of RTN3 to BACE1

Shi

et al. 2007

Journal of Biological Chemistry

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Reticulon 3 (RTN3) has recently been shown to modulate Alzheimer BACE1 activity and to play a role in the formation of dystrophic neurites present in Alzheimer brains. Despite the functional importance of this protein in Alzheimer disease pathogenesis, the functional correlation to the structural domain of RTN3 remained unclear. RTN3 has two long transmembrane domains, but its membrane topology was not known. We report here that the first transmembrane domain dictates membrane integration and its membrane topology. RTN3 adopts a -shape structure with two ends facing the cytosolic side. Subtle changes in RTN3 membrane topology can disrupt its binding to BACE1 and its inhibitory effects on BACE1 activity. Thus, the determination of RTN3 membrane topology may provide an important structural basis for our understanding of its cellular functions. Reticulon (RTN)2 proteins are a group of membrane-bound proteins that are largely localized in the endoplasmic reticulum (ER) (1, 2), perhaps more restricted to the tubular ER (3). RTNs exist in plants, fungi, and animals (4). In mammalian genomes, four independent reticulon genes, rtn1 to rtn4, have been identified to encode a large number of gene products (1, 2). All RTNs are composed of a variable length of N-terminal domain, which is completely divergent among RTN members, and a highly conserved RTN homology domain (RHD) that is unique to this family of proteins. Within the RHD, there exist two long stretches (28 -36 amino acids) of hydrophobic residues, and each is expected to embed RTNs in the membrane.The divergent N-terminal domain among RTNs potentially allows each RTN member to exert its specific function. RTN4, or its more popular acronym Nogo, is identified as a critical inhibitory molecule in axonal growth and regeneration (5, 6). Recently, reticulon proteins (including Nogo) have been shown to interact with ␤-secretase, also known as ␤-site APP-cleaving enzyme 1 (BACE1) (7-10), and this interaction negatively modulates the enzymatic activity of BACE1 (11-13). Among the RTNs, we have paid particular attention to RTN3 because of its strong inhibitory effects on BACE1 activity and high expression by neurons. RTN3 is localized not only in the ER compartment but also in the Golgi (11, 14), axons, dendrites, and growth cone (15). The localization of RTN3 in the neuritic region suggests its potential role in neuritic functions. Most recently, we have demonstrated that RTN3 also plays a role in the formation of dystrophic neurites in Alzheimer brains (15).Because of the important role of RTN3 in Alzheimer pathogenesis, we set out to study the structure and function of RTN3. We report herein that the first transmembrane stretch or domain (TM1) is important for the membrane insertion of RTN3. Disruption of the TM1 of RTN3 results in misfolding of the mutant protein and failure to integrate the protein into the membrane. We also show that RTN3 adopts a -shape membrane topology with both the N-and C-terminal ends facing the cytosolic side. Knowledge from this study will be...

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Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

Cited by 69 publications

References 52 publications

The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

The antiviral protein, viperin, localizes to lipid droplets via its N-terminal amphipathic α-helix

Murine Diacylglycerol Acyltransferase-2 (DGAT2) Can Catalyze Triacylglycerol Synthesis and Promote Lipid Droplet Formation Independent of Its Localization to the Endoplasmic Reticulum

The Membrane Topology of RTN3 and Its Effect on Binding of RTN3 to BACE1

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