Emery–Dreifuss muscular dystrophy mutations impair TRC40-mediated targeting of emerin to the inner nuclear membrane

Pfaff, Janine; Rivera‐Monroy, Jhon; Jamieson, Cara; Rajanala, Kalpana; Vilardi, Fabio; Schwappach, Blanche; Kehlenbach, Ralph H.

doi:10.1242/jcs.179333

Cited by 26 publications

(48 citation statements)

References 80 publications

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“…Next, we analyzed the steady-state levels of a subset of TA proteins by western blot and found that syntaxin 5 (Stx5), an essential SNARE involved in Golgi-to-ER trafficking, and emerin (EMD), a TA protein of the inner nuclear membrane37, were significantly less abundant compared to littermates not expressing the recombinase (Fig. 1e,f).…”

Section: Resultsmentioning

confidence: 99%

Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

Rivera‐Monroy

Musiol

Unthan-Fechner

et al. 2016

Sci Rep

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Tail-anchored (TA) proteins are post-translationally inserted into membranes. The TRC40 pathway targets TA proteins to the endoplasmic reticulum via a receptor comprised of WRB and CAML. TRC40 pathway clients have been identified using in vitro assays, however, the relevance of the TRC40 pathway in vivo remains unknown. We followed the fate of TA proteins in two tissue-specific WRB knockout mouse models and found that their dependence on the TRC40 pathway in vitro did not predict their reaction to receptor depletion in vivo. The SNARE syntaxin 5 (Stx5) was extremely sensitive to disruption of the TRC40 pathway. Screening yeast TA proteins with mammalian homologues, we show that the particular sensitivity of Stx5 is conserved, possibly due to aggregation propensity of its cytoplasmic domain. We establish that Stx5 is an autophagy target that is inefficiently membrane-targeted by alternative pathways. Our results highlight an intimate relationship between the TRC40 pathway and cellular proteostasis.

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Section: Resultsmentioning

confidence: 99%

Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

Rivera‐Monroy

Musiol

Unthan-Fechner

et al. 2016

Sci Rep

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“…Mutant forms of emerin show diminished transport to the inner nuclear membrane, and have been associated with decreased nuclear invagination and abnormalities in nuclear Ca ++ transients …”

Section: Pathophysiologymentioning

confidence: 99%

“…35,67 An exception is FHL1, a protein encoded by the gene of the same name, which localizes to the sarcomere and the sarcolemma; at the former, it contributes to sarcomere assembly. 38,39 Mutant forms of emerin show diminished transport to the inner nuclear membrane, 68 and have been associated with decreased nuclear invagination and abnormalities in nuclear Ca ++ transients. 69 In the case of LMNA mutations, and the associated effects on LMNA, there also appears to be an effect on myocytes and muscle regeneration, as these proteins are expressed in mature myocytes, skeletal muscle stem cells, and satellite cells.…”

Section: Pathophysiologymentioning

confidence: 99%

Emery‐Dreifuss muscular dystrophy

et al. 2019

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Emery‐Dreifuss muscular dystrophy (EDMD) is a rare muscular dystrophy, but is particularly important to diagnose due to frequent life‐threatening cardiac complications. EDMD classically presents with muscle weakness, early contractures, cardiac conduction abnormalities and cardiomyopathy, although the presence and severity of these manifestations vary by subtype and individual. Associated genes include EMD, LMNA, SYNE1, SYNE2, FHL1, TMEM43, SUN1, SUN2, and TTN, encoding emerin, lamin A/C, nesprin‐1, nesprin‐2, FHL1, LUMA, SUN1, SUN2, and titin, respectively. The Online Mendelian Inheritance in Man database recognizes subtypes 1 through 7, which captures most but not all of the associated genes. Genetic diagnosis is essential whenever available, but traditional diagnostic tools can help steer the evaluation toward EDMD and assist with interpretation of equivocal genetic test results. Management is primarily supportive, but it is important to monitor patients closely, especially for potential cardiac complications. There is a high potential for progress in the treatment of EDMD in the coming years.

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“…Emerin, loss of which causes X-linked Emery-Dreifuss muscular dystrophy [4][5][6][7][8], is one of the best-characterized proteins of the INM [9][10][11], although it was also described as a component of the ONM and the peripheral ER [12]. It is a tail-anchored protein with a single transmembrane domain close to its C-terminal end and we recently showed that emerin is inserted post-translationally into ER-membranes via the GET/TRC40 pathway [13]. Emerin has an N-terminal LEM-domain, which received its name from For SILAC, HeLa cells were labeled with heavy or light isotopes of arginine and lysine.…”

Section: Introductionmentioning

confidence: 99%

“…For this purpose, DMEM (high glucose) lacking glutamine, lysine and arginine (Thermo Fisher Scientific, Waltham, MA, USA) was supplemented with 10% (v/v) dialyzed FBS (Life technologies, Carlsbad, CA, USA), 6 mM L-glutamine (Life technologies), 100 U mL −1 penicillin and 100 µg mL −1 streptomycin. SILAC media was prepared as described [27], using 13 acids, respectively. To ensure sufficient incorporation of heavy amino acids, cells were passaged five to seven times in SILAC medium before the biotinylation experiment.…”

Section: Introductionmentioning

confidence: 99%

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling

Müller

James

Lenz

et al. 2020

Cells

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Emerin is one of the best characterized proteins of the inner nuclear membrane, but can also occur at the level of the endoplasmic reticulum. We now use enhanced ascorbate peroxidase 2 (APEX2) to probe the environment of emerin. APEX2 can be used as a genetic tag that produces short-lived yet highly reactive biotin species, allowing the modification of proteins that interact with or are in very close proximity to the tagged protein. Biotinylated proteins can be isolated using immobilized streptavidin and analyzed by mass spectrometry. As an alternative to the standard approach with a genetic fusion of APEX2 to emerin, we also used RAPIDS (rapamycin-and APEX-dependent identification of proteins by SILAC), a method with improved specificity, where the peroxidase interacts with the protein of interest (i.e., emerin) only upon addition of rapamycin to the cells. We compare these different approaches, which, together, identify well-known interaction partners of emerin like lamin A and the lamina associated polypeptide 1 (LAP1), as well as novel proximity partners.Cells 2020, 9, 605 2 of 18 three proteins containing this characteristic feature (Lap2, emerin and Man1; [14]). Major binding partners of emerin are A-and also B-type lamins [6]. Indeed, the localization of emerin at the nuclear envelope was shown to depend on lamin A [15]. Hence, retention of emerin upon binding to lamins plays an important role in INM-targeting. Besides lamins, a number of emerin interaction partners have been described, for example BAF (barrier-to-autointegration factor (BANF1) [16], nesprin-1α [17] and HDAC3 [18]; for review see [9]).For proteins of the INM, the identification of interacting proteins is particularly challenging, because conditions that are typically used in, for example, co-immunoprecipitation approaches, may not lead to complete solubilization of NE-structures. Genetic approaches like the yeast-two-hybrid method, on the other hand, detect interactions under rather non-physiological conditions. Neither approach addresses the native subcellular localization of a protein of interest. To overcome these problems, several methods have been developed that use proximity-based labeling, typically introducing biotin into unknown proteins, allowing subsequent affinity capture with immobilized streptavidin and analysis by mass-spectrometry (for review see [19]). One approach termed BioID uses a biotin ligase from E. coli for biotinylation of proteins [20]. As an alternative, ascorbate peroxidase (APEX), an enzyme that generates radicals from biotin phenol in the presence of H 2 O 2 was introduced [21]. Modification of proteins occurs within a range of~20 nm of the enzyme, which can be genetically fused to the Nor C-terminus of proteins of interest for probing their direct environment and identifying potential interaction partners [22][23][24][25]. An improved version of APEX, APEX2, is far more active than the original enzyme [26]. Very recently, we devised a method where APEX2 is not directly fused, but instead targeted to a protei...

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Emery–Dreifuss muscular dystrophy mutations impair TRC40-mediated targeting of emerin to the inner nuclear membrane

Cited by 26 publications

References 80 publications

Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

Mice lacking WRB reveal differential biogenesis requirements of tail-anchored proteins in vivo

Emery‐Dreifuss muscular dystrophy

Probing the Environment of Emerin by Enhanced Ascorbate Peroxidase 2 (APEX2)-Mediated Proximity Labeling

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