A calreticulin-dependent nuclear export signal is involved in the regulation of liver receptor homologue-1 protein folding.
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.
Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei 100, Taiwan.
Feng-Ming
FM
Yang
Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei 100, Taiwan.
Shan-Jung
SJ
Feng
Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei 100, Taiwan.
Tsai-Chun
TC
Lai
Graduate Institute of Physiology, National Taiwan University College of Medicine, Taipei 100, Taiwan mengchun@ntu.edu.tw.
Meng-Chun
MC
Hu
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000818:Animals; D019556:COS Cells; D064032:Calbindin 2 / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D049790:Nuclear Export Signals / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*
C576511:Calb2 protein, mouse; D064032:Calbindin 2; D018840:HSP70 Heat-Shock Proteins; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; C431936:Nr5a2 protein, mouse; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear
aggresome; autophagy; calreticulin; heat shock protein 70; liver receptor homologue-1 (LRH-1); nuclear export signal