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

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Abstract

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF–GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.

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.

    1

    Present address: Department of Geriatric Research, National Institute for Longevity Sciences, Obu, Aichi 474-8522, Japan.

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