Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

Snapp, Erik L.; Sharma, Ajay; Lippincott‐Schwartz, Jennifer; Hegde, Ramanujan S.

doi:10.1073/pnas.0510657103

Cited by 114 publications

(124 citation statements)

References 45 publications

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“…Failure of CDDP to induce CRT redistribution from the ER lumen to the cell surface To monitor the redistribution of CRT from the ER lumen to peripheral locations close to the plasma membrane, we generated U2OS cells that stably express a CRT-green fluorescent protein (GFP) fusion protein (Snapp et al, 2006). Control experiments revealed that this protein was located in the ER lumen, in which most of the endogenous CRT resides (not shown).…”

Section: Resultsmentioning

confidence: 99%

Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress

et al. 2010

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In contrast to other cytotoxic agents including anthracyclins and oxaliplatin (OXP), cisplatin (CDDP) fails to induce immunogenic tumor cell death that would allow to stimulate an anticancer immune response and hence to amplify its therapeutic efficacy. This failure to induce immunogenic cell death can be attributed to CDDP's incapacity to elicit the translocation of calreticulin (CRT) from the lumen of the endoplasmic reticulum (ER) to the cell surface. Here, we show that, in contrast to OXP, CDDP is unable to activate the protein kinase-like ER kinase (PERK)-dependent phosphorylation of the eukaryotic translation initiation factor 2a (eIF2a). Accordingly, CDDP also failed to stimulate the formation of stress granules and macroautophagy, two processes that only occur after eIF2a phosphorylation. Using a screening method that monitors the voyage of CRT from the ER lumen to the cell surface, we identified thapsigargin (THAPS), an inhibitor of the sarco/ER Ca 2 þ -ATPase as a molecule that on its own does not stimulate CRT exposure, yet endows CDDP with the capacity to do so. The combination of ER stress inducers (such as THAPS or tunicamycin) and CDDP effectively induced the translocation of CRT to the plasma membrane, as well as immunogenic cell death, although ER stress or CDDP alone was insufficient to induce CRT exposure and immunogenic cell death. Altogether, our results underscore the contribution of the ER stress response to the immunogenicity of cell death.

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Section: Resultsmentioning

confidence: 99%

Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress

et al. 2010

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“…FL-DGAT2 and FL-⌬122-388 displayed a typical ER staining pattern and co-localized with the ER marker, ER-GFP (Fig. 3A) (30). When both transmembrane domains were deleted from DGAT2 (FL-⌬66 -115), DGAT2 was no longer in the ER and did not co-localize with ER-GFP (Fig.…”

Section: Dgat2 Is a Multimericmentioning

confidence: 94%

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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“…LULL1-mCherry was made by excising LULL1 from EGFP-N1 using HindIII and EcoRI restriction sites and ligating into pmCherry-N1 (Clontech, Mountain View, CA). ER-RFP was from (Snapp et al, 2006). Sun2-GFP was from Hodzic et al (2004).…”

Section: Plasmidsmentioning

confidence: 99%

“…This, together with the fact that ⌬GAG-TorA (and in some cell types, also wild-type TorA) is known to be intrinsically enriched in the NE ( Figure 1A; Gonzalez-Alegre and Paulson, 2004; Goodchild and Dauer, 2004;Naismith et al, 2004), led us to hypothesize that distribution of TorA between ER and NE may normally be controlled by interaction with endogenous LULL1. To explore this possibility, we used RNA interference (RNAi) to deplete LULL1 ( Figure 5A) and compared the localization of ⌬GAG-TorA-mGFP to that of a cotransfected ER lumenal marker consisting of a prolactin signal sequence and a KDEL ER-retrieval sequence fused to mRFP (ER-RFP; Snapp et al, 2006). Representative confocal sections show that ⌬GAG-TorA-mGFP is more concentrated around the nucleus than is ER-RFP in U2OS cells ( Figure 5B), but not in LULL1 knockdown cells ( Figure 5C).…”

Section: Removing Lull1 Reduces Enrichment Of ⌬Gag-tora In the Nucleamentioning

confidence: 99%

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

Heyden

Naismith

Snapp

et al. 2009

MBoC

Self Cite

115

162

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TorsinA (TorA) is an AAA؉ ATPase in the endoplasmic reticulum (ER) lumen that is mutated in early onset DYT1 dystonia. TorA is an essential protein in mice and is thought to function in the nuclear envelope (NE) despite localizing throughout the ER. Here, we report that transient interaction of TorA with the ER membrane protein LULL1 targets TorA to the NE. FRAP and Blue Native PAGE indicate that TorA is a stable, slowly diffusing oligomer in either the absence or presence of LULL1. Increasing LULL1 expression redistributes both wild-type and disease-mutant TorA to the NE, while decreasing LULL1 with shRNAs eliminates intrinsic enrichment of disease-mutant TorA in the NE. When concentrated in the NE, TorA displaces the nuclear membrane proteins Sun2, nesprin-2G, and nesprin-3 while leaving nuclear pores and Sun1 unchanged. Wild-type TorA also induces changes in NE membrane structure. Because SUN proteins interact with nesprins to connect nucleus and cytoskeleton, these effects suggest a new role for TorA in modulating complexes that traverse the NE. Importantly, once concentrated in the NE, disease-mutant TorA displaces Sun2 with reduced efficiency and does not change NE membrane structure. Together, our data suggest that LULL1 regulates the distribution and activity of TorA within the ER and NE lumen and reveal functional defects in the mutant protein responsible for DYT1 dystonia.

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Monitoring chaperone engagement of substrates in the endoplasmic reticulum of live cells

Cited by 114 publications

References 45 publications

Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress

Restoration of the immunogenicity of cisplatin-induced cancer cell death by endoplasmic reticulum stress

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

LULL1 Retargets TorsinA to the Nuclear Envelope Revealing an Activity That Is Impaired by theDYT1Dystonia Mutation

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