Interaction of BIG2, a brefeldin A-inhibited guanine nucleotide-exchange protein, with exocyst protein Exo70.
Guanine nucleotide-exchange proteins activate ADP-ribosylation factors by accelerating the replacement of bound GDP with GTP. Mammalian brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are important activators of ADP-ribosylation factors for vesicular trafficking. To identify proteins that interact with BIG2, we used cDNA constructs encoding BIG2 sequences in a yeast two-hybrid screen of a human heart library. Clone p2-5-3, encoding a form of human exocyst protein Exo70, interacted with BIG2 amino acids 1-643 and 1-832, but not 644-832, which was confirmed by coimmunoprecipitation of in vitro-translated BIG2 N-terminal segments and 2-5-3. By immunofluorescence microscopy, endogenous BIG2 and Exo70 in HepG2 cells were visualized at Golgi membranes and apparently at the microtubule-organizing center (MTOC). Both were identified in purified centrosomes. Immunoreactive Exo70 and BIG2 partially or completely overlapped with gamma-tubulin at the MTOC in cells inspected by confocal microscopy. In cells incubated with brefeldin A, most of the BIG2, Exo70, and trans-Golgi protein p230 were widely dispersed from their perinuclear concentrations, but small amounts always remained, apparently at the MTOC. After disruption of microtubules with nocodazole, BIG2 and Exo70 were widely distributed in cells and remained only partially colocalized with p230, BIG2 more so than Exo70. We conclude that in HepG2 cells BIG2 and Exo70 interact in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane, consistent with a functional association in both early and late stages of vesicular trafficking.
Xu
Kai-Feng
KF
Pulmonary-Critical Care Medicine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Shen
Xiaoyan
X
Li
Hewang
H
Pacheco-Rodriguez
Gustavo
G
Moss
Joel
J
Vaughan
Martha
M
D000595:Amino Acid Sequence; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D005089:Exocytosis; D020662:Guanine Nucleotide Exchange Factors / Q000032:analysis / Q000502:physiology*; D006801:Humans; D022101:Microtubule-Organizing Center / Q000737:chemistry; D008969:Molecular Sequence Data; D015739:Nocodazole / Q000494:pharmacology; D020798:Two-Hybrid System Techniques; D021601:trans-Golgi Network / Q000737:chemistry
C119088:ARFGEF2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D020126:Brefeldin A; D015739:Nocodazole