Dominant-negative mutant of BIG2, an ARF–guanine nucleotide exchange factor, specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins☆
Section snippets
Materials and methods
Plasmid construction. An E738K mutation was introduced into the human BIG2 cDNA [14] by a PCR-based strategy. The wild-type or mutated BIG2 cDNA was subcloned into pcDNA4-HAN [15], which is an expression vector for N-terminally hemagglutinin (HA)-tagged protein.
Antibodies. For production of polyclonal antisera to GGA, glutathione S-transferase fused to a portion of human GGA1 (amino acids 297–493) was expressed in Escherichia Coli JM109 cells and purified by glutathione–Sepharose affinity
Results
To construct a potential dominant-negative mutant of BIG2, we replaced a Glu residue in its Sec7 domain by a Lys residue (E738K). Such a type of mutation was first found in the deficient emb30 allele (E658K) in Arabidopsis[18] and was later shown to result in loss of the GEF activity in ARNO(E156K) [19], [20]. Furthermore, ARNO(E156K) was shown to form a stable complex with GDP-bound ARF without inducing the release of GDP [21]. Because the stable complex between ARF and a GEF is also formed in
Discussion
ARF–GEFs, especially high molecular ones, are key regulators for the formation of coated carrier vesicles. The present study extends our previous study [14] and shows that BIG2, an ARF–GEF, is involved in recruitment of the AP-1 complex and GGA onto TGN membranes but not that of the COPI complex onto the cis-Golgi. This conclusion is based on experiments using a dominant-negative mutant of BIG2, BIG2(E738K). Although such Glu-to-Lys mutations in the Sec7 catalytic domains are known to abolish
Acknowledgements
We thank Dr. Minoru Fukuda for providing anti-TGN46 antiserum. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Japan Society for Promotion of Science, and the University of Tsukuba Research Projects.
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Abbreviations: ARF, ADP-ribosylation factor; BFA, brefeldin A; GEF, guanine nucleotide exchange factor; GGA, Golgi-localizing, γ-adaptin ear homology domain, ARF-binding protein; HA, hemagglutinin; MPR, mannose 6-phosphate receptor; TGN, trans-Golgi network.
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Present address: Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi 474-8522, Japan.