WRB and CAML Are Necessary and Sufficient to Mediate Tail-Anchored Protein Targeting to the ER Membrane

Vilardi, Fabio; Stephan, Milena; Clancy, Anne; Janshoff, Andreas; Schwappach, Blanche

doi:10.1371/journal.pone.0085033

Cited by 67 publications

(77 citation statements)

References 30 publications

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“…Because CAML and WRB are recognized as the mammalian TRC40 receptor (22,23,29), we investigated whether immunoadsorption of microsomal TRC40 caused depletion of these two polypeptides. We followed the distribution of CAML, WRB, and TRC40 between the DBC soluble and insoluble MR fractions ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Because structural or functional homologues of all components of the Get system are present in mammals and some of the central components of the pathway are interchangeable between yeast and higher eukaryotes (16,22), it is thought that the entire Get pathway is conserved in higher eukaryotes (for review, see Refs. 3 and 4).…”

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confidence: 99%

“…However, pulldown experiments with TRC40 as bait identified CAML (calcium modulating cyclophilin ligand) as a TRC40-and WRB-interacting protein (23). siRNA-mediated depletion of CAML or WRB partially inhibits insertion of in vitro translated TA proteins into the ER of semiintact cells (23), and the WRB-CAML complex can restore function of the Get system in yeast cells deleted for the Get1/2 complex (22). Thus, CAML is considered to be the functional equivalent of Get2.…”

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confidence: 99%

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Tail-anchored Protein Insertion in Mammals

Colombo

Cardani

Maroli

et al. 2016

Journal of Biological Chemistry

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The GET (guided entry of tail-anchored proteins)/TRC (transmembrane recognition complex) pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function similarly in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1. Get3/TRC40 function requires an ER receptor, which in yeast consists of the Get1/ Get2 heterotetramer and in mammals of the WRB protein (tryptophan-rich basic protein), homologous to yeast Get1, in combination with CAML (calcium-modulating cyclophilin ligand), which is not homologous to Get2. To better characterize the mammalian receptor, we investigated the role of endogenous WRB and CAML in tail-anchored protein insertion as well as their association, concentration, and stoichiometry in rat liver microsomes and cultured cells. Functional proteoliposomes, reconstituted from a microsomal detergent extract, lost their activity when made with an extract depleted of TRC40-associated proteins or of CAML itself, whereas in vitro synthesized CAML and WRB together were sufficient to confer insertion competence to liposomes. CAML was found to be in ϳ5-fold excess over WRB, and alteration of this ratio did not inhibit insertion. Depletion of each subunit affected the levels of the other one; in the case of CAML silencing, this effect was attributable to destabilization of the WRB transcript and not of WRB protein itself. These results reveal unanticipated complexity in the mutual regulation of the TRC40 receptor subunits and raise the question as to the role of the excess CAML in the mammalian ER.Whereas most membrane proteins are targeted to the endoplasmic reticulum (ER) 3 by the signal recognition particle-dependent co-translational mechanism (1, 2), tail-anchored (TA) membrane proteins are inserted into all their target membranes, including the ER, by post-translational pathways (for review see Refs. 3-5). The inability of TA proteins to utilize the co-translational route is due to the location of their sole targeting determinant, the transmembrane domain (TMD), at their extreme C terminus, such that it becomes accessible to cytosolic targeting factors only after release of the completed polypeptide chain from the ribosome. Because TA proteins are numerous (6), play fundamental physiological roles (e.g. as SNAREs and apoptosis regulating factors; Ref.3), and are present in all domains of life (7), a great deal of research has been dedicated to the elucidation of the mechanisms by which they reach and insert into their target membranes. On the basis of studies in cell-free mammalian systems (8, 9) and of in vitro and in vivo investigations in yeast (10), a novel system operating in the delivery of TA proteins to the ER membrane was identified and extensively characterized (for review, see Refs. 11 and 12). This system is centered around a cytosolic P-type ATPase, named Get3 (guided entry of tailanchored proteins) in yeast and TRC40 (transmembrane domain recognition complex subunit of 40...

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Section: Resultsmentioning

confidence: 99%

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Tail-anchored Protein Insertion in Mammals

Colombo

Cardani

Maroli

et al. 2016

Journal of Biological Chemistry

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“…Important players are TRC40 [transmembrane domain recognition complex protein of 40 kDa, also known as ASNA1; guided entry of tail-anchored proteins 3 (Get3) in yeast], a protein that associates with the hydrophobic stretch of amino acids at the C-terminus of tail-anchored proteins (Favaloro et al, 2008;Stefanovic and Hegde, 2007), WRB (tryptophan-rich basic protein; Get1 in yeast) (Vilardi et al, 2011) and the mammalian-specific protein CAML [Ca 2+ -modulating cyclophilin ligand, also known as CAMLG (Yamamoto and Sakisaka, 2012)]. Together, WRB and CAML function as the TRC40 receptor at the ER membrane (Vilardi et al, 2011(Vilardi et al, , 2014Yamamoto and Sakisaka, 2012). Furthermore, chaperone-like components, such as SGTA and BAG6, capture the C-terminal transmembrane domains as they emerge from the ribosome and then deliver tail-anchored proteins to TRC40 (Leznicki et al, 2010;Mariappan et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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

Pfaff

Rivera‐Monroy

Jamieson

et al. 2016

Journal of Cell Science

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Emerin is a tail-anchored protein that is found predominantly at the inner nuclear membrane (INM), where it associates with components of the nuclear lamina. Mutations in the emerin gene cause EmeryDreifuss muscular dystrophy (EDMD), an X-linked recessive disease. Here, we report that the TRC40/GET pathway for post-translational insertion of tail-anchored proteins into membranes is involved in emerin-trafficking. Using proximity ligation assays, we show that emerin interacts with TRC40 in situ. Emerin expressed in bacteria or in a cell-free lysate was inserted into microsomal membranes in an ATP-and TRC40-dependent manner. Dominant-negative fragments of the TRC40-receptor proteins WRB and CAML (also known as CAMLG) inhibited membrane insertion. A rapamycin-based dimerization assay revealed correct transport of wild-type emerin to the INM, whereas TRC40-binding, membrane integration and INMtargeting of emerin mutant proteins that occur in EDMD was disturbed. Our results suggest that the mode of membrane integration contributes to correct targeting of emerin to the INM.

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“…S1). Studies on the yeast TA targeting pathway (7,(11)(12)(13) and the mammalian TA targeting (6,(14)(15)(16) and ubiquitination (9,10) reactions suggest that six core factors constitute a minimal mammalian triage system amenable to complete reconstitution.…”

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Mechanistic basis for a molecular triage reaction

Shao

Rodrigo-Brenni

Kivlen

et al. 2017

Science

125

203

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Newly synthesized proteins are triaged between biosynthesis and degradation to maintain cellular homeostasis, but the decision-making mechanisms are unclear. Here, we reconstitute the core reactions for membrane targeting and ubiquitination of nascent tail-anchored membrane proteins to understand how their fate is determined. The central six-component triage system is divided into an uncommitted client-SGTA complex, a self-sufficient targeting module, and an embedded but self-sufficient quality control module. Client-SGTA engagement of the targeting module induces rapid, private, and committed client transfer to TRC40 for successful biosynthesis. Commitment to ubiquitination is dictated primarily by comparatively slower client dissociation from SGTA and non-private capture by the Bag6 subunit of the quality control module. This provides a paradigm for how priority and time are encoded within a multi-chaperone triage system.Protein biosynthesis and quality control pathways are precisely balanced to provide nascent proteins an initial opportunity to mature, while favoring degradation over time (1-3). Deviations from this balance lead to premature degradation of normal maturation intermediates or persistence of misfolded proteins, either of which can cause disease (2,4,5). Although accurate triage between biosynthesis and degradation is essential for maintaining protein homeostasis, a mechanistic understanding of protein triage for any pathway has been hampered by the lack of a fully reconstituted system that faithfully recapitulates both the biosynthetic and quality control outcomes of a nascent protein.To achieve this goal, we chose the pathway of tail-anchored (TA) membrane protein insertion as a model. TA proteins engage a conserved and well-defined pathway for targeting and insertion at the ER (6-8), which in mammals is monitored by an embedded quality control (QC) pathway (9, 10) to degrade products that fail targeting ( fig. S1). Studies on the yeast TA targeting pathway (7,(11)(12)(13) and the mammalian TA targeting (6, 14-16) and ubiquitination (9, 10) reactions suggest that six core factors constitute a minimal mammalian triage system amenable to complete reconstitution.Three of the factors (SGTA, Bag6, and TRC40) can recognize and shield the transmembrane domain (TMD) of a TA protein client (6,(16)(17)(18) Analyses with purified factors rigorously established the sufficiency of these modules. In these experiments, radiolabeled TA protein is synthesized with purified E. coli translation factors supplemented with the desired TMD-binding chaperone ( fig. S5). This orthogonal system produces homogeneous TA-chaperone complexes that can be used for downstream functional assays (24). Isolated TA-TRC40 complex was competent for ER targeting and insertion ( Fig. 2A), but not ubiquitination by RNF126 ( fig. S6). By contrast, the TA-Bag6 complex could not mediate insertion (data not shown), but allowed ubiquitination via RNF126 recruitment to Bag6's N-terminal UBL domain (Fig. 2B).The TA-SGTA complex in iso...

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WRB and CAML Are Necessary and Sufficient to Mediate Tail-Anchored Protein Targeting to the ER Membrane

Cited by 67 publications

References 30 publications

Tail-anchored Protein Insertion in Mammals

Tail-anchored Protein Insertion in Mammals

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

Mechanistic basis for a molecular triage reaction

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