title: Tail-anchored Protein Insertion in Mammals: FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR.
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.
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and s.colombo@in.cnr.it.
authors.forename: Sara Francesca
authors.identifier: 0000-0001-9714-3961
authors.initials: SF
authors.lastname: Colombo
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and.
authors.forename: Silvia
authors.initials: S
authors.lastname: Cardani
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and.
authors.forename: Annalisa
authors.initials: A
authors.lastname: Maroli
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and.
authors.forename: Adriana
authors.initials: A
authors.lastname: Vitiello
authors.affiliation: IFOM, the FIRC Institute for Molecular Oncology Foundation, Milan, Italy 20100 and.
authors.forename: Paolo
authors.initials: P
authors.lastname: Soffientini
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and.
authors.forename: Arianna
authors.initials: A
authors.lastname: Crespi
authors.affiliation: Mayo Clinic, Rochester, Minnesota 55905.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Bram
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and.
authors.forename: Roberta
authors.identifier: 0000-0001-5396-5429
authors.initials: R
authors.lastname: Benfante
authors.affiliation: From the CNR Institute of Neuroscience and BIOMETRA Department, Università degli Studi di Milano and n.borgese@in.cnr.it.
authors.forename: Nica
authors.identifier: 0000-0002-7537-3974
authors.initials: N
authors.lastname: Borgese
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D053501:Arsenite Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D004314:Down Syndrome / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005786:Gene Expression Regulation; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits; D021381:Protein Transport; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C089404:CAMLG protein, human; C490313:Camlg protein, rat; C111546:GET1 protein, human; C489667:GET3 protein, human; D020662:Guanine Nucleotide Exchange Factors; D009687:Nuclear Proteins; D021122:Protein Subunits; D011510:Proteolipids; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; D053501:Arsenite Transporting ATPases; C529048:Get3 protein, S cerevisiae
keywords: Down syndrome; Proteoliposomes; endoplasmic reticulum (ER); mRNA stability; membrane biogenesis; membrane protein; membrane reconstitution; post-translational insertion; protein translocation