A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding

Biochem J. 2015 Oct 15;471(2):199-209. doi: 10.1042/BJ20150252. Epub 2015 Aug 12.

Abstract

As an orphan member of the nuclear receptor family, liver receptor homologue-1 (LRH-1) controls a tremendous range of transcriptional programmes that are essential for metabolism and hormone synthesis. Our previous studies have shown that nuclear localization of the LRH-1 protein is mediated by two nuclear localization signals (NLSs) that are karyopherin/importin-dependent. It is unclear whether LRH-1 can be actively exported from the nucleus to the cytoplasm. In the present study, we describe a nuclear export domain containing two leucine-rich motifs [named nuclear export signal (NES)1 and NES2] within the ligand-binding domain (LBD). Mutation of leucine residues in NES1 or NES2 abolished nuclear export, indicating that both NES1 and NES2 motifs are essential for full nuclear export activity. This NES-mediated nuclear export was insensitive to the chromosomal region maintenance 1 (CRM1) inhibitor leptomycin B (LMB) or to CRM1 knockdown. However, knockdown of calreticulin (CRT) prevented NES-mediated nuclear export. Furthermore, our data show that CRT interacts with LRH-1 and is involved in the nuclear export of LRH-1. With full-length LRH-1, mutation of NES1 led to perinuclear accumulation of the mutant protein. Immunofluorescence analysis showed that these perinuclear aggregates were co-localized with the centrosome marker, microtubule-associated protein 1 light chain 3 (LC3), ubiquitin and heat shock protein 70 (Hsp70), indicating that the mutant was misfolded and sequestered into aggresome-like structures via the autophagic clearance pathway. Our study demonstrates for the first time that LRH-1 has a CRT-dependent NES which is not only required for cytoplasmic trafficking, but also essential for correct protein folding to avoid misfolding-induced aggregation.

Keywords: aggresome; autophagy; calreticulin; heat shock protein 70; liver receptor homologue-1 (LRH-1); nuclear export signal.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus / physiology
  • Animals
  • COS Cells
  • Calbindin 2 / genetics
  • Calbindin 2 / metabolism*
  • Cell Nucleus / genetics
  • Cell Nucleus / metabolism*
  • Chlorocebus aethiops
  • Cytoplasm / genetics
  • Cytoplasm / metabolism*
  • HSP70 Heat-Shock Proteins / genetics
  • HSP70 Heat-Shock Proteins / metabolism
  • Mice
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism
  • Nuclear Export Signals / physiology*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*

Substances

  • Calb2 protein, mouse
  • Calbindin 2
  • HSP70 Heat-Shock Proteins
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Nr5a2 protein, mouse
  • Nuclear Export Signals
  • Receptors, Cytoplasmic and Nuclear