Tail-anchored protein insertion in mammals function and reciprocal interactions of the two subunits of the trc40receptor

Sara Francesca Colombo, Silvia Cardani, Annalisa Maroli, Adriana Vitiello, Paolo Soffientini, Arianna Crespi, Richard J Bram, Roberta Benfante, Nica Borgese

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
Pages (from-to)15292-15306
Number of pages15
JournalJournal of Biological Chemistry
Volume291
Issue number29
DOIs
StatePublished - Jul 15 2016

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Cyclophilins
Mammals
Tail
Ligands
Calcium
Endoplasmic Reticulum
Proteins
Yeast
Yeasts
Liver Microsomes
Protein Transport
Liposomes
Tryptophan
Stoichiometry
Detergents
Liver
Mental Competency
Adenosine Triphosphatases
Rats
Cultured Cells

ASJC Scopus subject areas

  • Biochemistry
  • Medicine(all)
  • Molecular Biology
  • Cell Biology

Cite this

Colombo, S. F., Cardani, S., Maroli, A., Vitiello, A., Soffientini, P., Crespi, A., ... Borgese, N. (2016). Tail-anchored protein insertion in mammals function and reciprocal interactions of the two subunits of the trc40receptor. Journal of Biological Chemistry, 291(29), 15292-15306. https://doi.org/10.1074/jbc.M115.707752

Tail-anchored protein insertion in mammals function and reciprocal interactions of the two subunits of the trc40receptor. / Colombo, Sara Francesca; Cardani, Silvia; Maroli, Annalisa; Vitiello, Adriana; Soffientini, Paolo; Crespi, Arianna; Bram, Richard J; Benfante, Roberta; Borgese, Nica.

In: Journal of Biological Chemistry, Vol. 291, No. 29, 15.07.2016, p. 15292-15306.

Research output: Contribution to journalArticle

Colombo, SF, Cardani, S, Maroli, A, Vitiello, A, Soffientini, P, Crespi, A, Bram, RJ, Benfante, R & Borgese, N 2016, 'Tail-anchored protein insertion in mammals function and reciprocal interactions of the two subunits of the trc40receptor', Journal of Biological Chemistry, vol. 291, no. 29, pp. 15292-15306. https://doi.org/10.1074/jbc.M115.707752
Colombo, Sara Francesca ; Cardani, Silvia ; Maroli, Annalisa ; Vitiello, Adriana ; Soffientini, Paolo ; Crespi, Arianna ; Bram, Richard J ; Benfante, Roberta ; Borgese, Nica. / Tail-anchored protein insertion in mammals function and reciprocal interactions of the two subunits of the trc40receptor. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 29. pp. 15292-15306.
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