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Brefeldin A‐inhibited guanine nucleotide‐exchange protein 1 (BIG1) associates with nucleolin and U3 snoRNA in HepG2 cell nuclei
Author(s) -
Padilla Philip Ian,
Uhart Marina,
Peculis Brenda,
Moss Joel,
Vaughan Martha
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.816.3
Subject(s) - nucleolin , brefeldin a , microbiology and biotechnology , nucleolus , biology , immunoprecipitation , chemistry , golgi apparatus , endoplasmic reticulum , biochemistry , cytoplasm , gene
BIG1, a brefeldin A‐inhibited guanine nucleotide‐exchange protein, activates class I ADP‐ribosylation factors (ARF1–3) by catalyzing replacement of bound GDP by GTP, an action critical in secretory transport from endoplasmic reticulum to cis‐Golgi. Our earlier report that BIG1 concentrated in nucleoli of serum‐starved HepG2 cells (Padilla et al., 2004) prompted us to identify molecules associated with BIG1 in the nucleolus. Antibodies against BIG1 co‐precipitated nucleolin (No, an abundant nucleolar protein) from nuclei; likewise, antibodies against No would co‐precipitate BIG1. Incubation of nuclei with RNAse or DNAse abolished co‐immunoprecipitation of BIG1 and No indicating the interaction depended on nucleic acids. Analysis of small nucleolar (sno)RNAs immunoprecipitated with BIG1 and No from nuclei revealed predominant bands of ~210 and ~70 bases; hybridization of the larger with U3‐specific oligonucleotides confirmed its identity. Cloning of U3 snoRNA cDNAs from the material precipitated by antibodies against BIG1 or No yielded identical nucleotide sequences. Continuing characterization of the mechanism and function of BIG1 accumulation in nucleoli of serum‐deprived cells may facilitate their comparison with those of the rapid nuclear accumulation of BIG1 that resulted from protein kinase A‐catalyzed phosphorylation of BIG1 Ser 883. Supported by Intramural Research Program, NHLBI, NIH.