Abstract
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 ∼5-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.
Highlights
The GET/TRC pathway for tail-anchored protein targeting to the endoplasmic reticulum (ER) has been characterized in detail in yeast and is thought to function in mammals, where the orthologue of the central ATPase, Get3, is known as TRC40 or Asna1
In this study we have analyzed the role of the TRC40 receptor subunits, CAML and WRB, in TA protein insertion into the ER and shown that the two subunits, synthesized in vitro, are sufficient to confer insertion competence to phospholipid vesicles
We find that a stoichiometric excess of one subunit over the other does not affect TA protein insertion and, in agreement, that endogenous CAML is present in an ϳ5-fold molar excess over endogenous WRB both in rat liver microsomes and in cultured cells
Summary
Plasmids—pGEM4 plasmids coding for b5 with a C-terminal opsin tag (b5-ops28) and for b5 with the TMD of synaptobrevin 2 and a C-terminal opsin tag (b5-TMSyb2-ops28), both under the SP6 promoter, have been described in previous publications [27, 28]. The purified recombinant proteins were used as standards for the determination of WRB and CAML concentration in microsomes, cultured cell lysates, and in vitro translated samples. A microsomal detergent extract was prepared by incubating the MR (at ϳ8 mg protein/ml) with 0.8% Deoxy Big Chap (DBC) for 30 min on ice. The sample was centrifuged at 100,000 ϫ g for 1 h, and the supernatant was used for reconstitution experiments. Somal protein or 70 l of wheat germ extract containing in vitro translated WRB or CAML were incubated in a total volume of 200 l containing 1 mg of PC ϩ NBD-PC, salts (0.8 M KOAc, 50 mM Hepes-Kϩ, pH 7.5, 5 mM Mg(OAc)2), 15% glycerol, 1% DBC, and 215 g of Bio-Beads.
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