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Dhirendra P. Singh, Yoshihiro Takamura, Bhavana Chhunchha, Nigar Fatma, Biju Bhargavan, Eri Kubo; Characterization of Lens Epithelium Derived-Protein/132, a Novel Guanine Nucleotide Exchange Factor for ARFs, and Its Role in ER and Golgi Homeostasis. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1527.
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© ARVO (1962-2015); The Authors (2016-present)
Cellular stress from environmental insults that perturb function of the endoplasmic reticulum (ER stress) has been implicated in diseases. Using expression cloning from a cDNA library prepared from human lens epithelium cells (hLECs), we identified a novel 132kD protein with guanine exchange factor (GEF) motifs for activation of ADP-ribosylation factor (ARF). We characterized the functional motif, and studied the contribution of LEDP/132 expression levels in maintaining Golgi and ER homeostasis and postponing ER-initiated death signaling.
LEDP/132 cDNA was sequenced and protein coding region was determined. A full length of cDNA was cloned into pGEX-5x-3 and pGFP expression vectors. Bioinformatics-based deletion mutants linked to GFP/GST were engineered to determine functional motifs, and localization was ascertained using cell organelle specific markers and immunocytochemistry with LEDP/132 and organelle specific antibodies. ER stress was induced by UVB exposure, H2O2 and tunicamycin in cells over-or under-expressing LEDP/132. Coprecipitation/pull down assay was used for the interaction of LEDP/132 and ARF. ARF activation assay was done by commercial kit. MTS plus TUNEL and Brefeldin A (BFA) assays were used to define cell death and LEDP/132 activity, respectively.
LEDP/132, was derived from hLECs and has two ARF-GEF motifs (amino acid [aa] 244/377 and 674/834), three coil-coil with ER (aa,525KDEL528) and Golgi (aa,136RRXXKKXXRR430) retention motifs. Immunohistochemical and cotransfections studies revealed that LEDP/132 colocalizes with Golgi, ER and transporting vesicles. Deletion mutants of LEDP/132 revealed that N-terminal (aa, 1/610) and C-terminal (aa,610/925) both are essential for Golgi-ER localization. BFA treatment had no effect on Golgi structure integrity in cells overexpressing LEDP/132. ARF activation assay showed the LEDP/132 mediated- activation of ARF1. Cells overexpressing LEDP/132 optimized the expression of Bip (a marker of ER stress), and gained resistance against ER-stressors.
LEDP/132 has functional ARF-GEF motifs which protects hLECs from ER stress-evoked deleterious signaling by ARF1 activation-mediated maintenance of ER-Golgi homeostasis. To our knowledge this is the first description of LEDP/132 as a new member of the ER-Golgi associated BFA-resistant ARF-GEF protein family.
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