Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Oct 21;286(42):36898-906.
doi: 10.1074/jbc.M111.230631. Epub 2011 Aug 2.

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation

Jason Lowery  1 , Tomasz Szul, Jayaraman Seetharaman, Xiaoying Jian, Min Su, Farhad Forouhar, Rong Xiao, Thomas B Acton, Gaetano T Montelione, Helen Lin, John W Wright, Eunjoo Lee, Zoe G Holloway, Paul A Randazzo, Liang Tong, Elizabeth Sztul
Affiliations

Novel C-terminal motif within Sec7 domain of guanine nucleotide exchange factors regulates ADP-ribosylation factor (ARF) binding and activation

Jason Lowery et al. J Biol Chem. .

Abstract

ADP-ribosylation factors (ARFs) and their activating guanine nucleotide exchange factors (GEFs) play key roles in membrane traffic and signaling. All ARF GEFs share a ∼200-residue Sec7 domain (Sec7d) that alone catalyzes the GDP to GTP exchange that activates ARF. We determined the crystal structure of human BIG2 Sec7d. A C-terminal loop immediately following helix J (loop>J) was predicted to form contacts with helix H and the switch I region of the cognate ARF, suggesting that loop>J may participate in the catalytic reaction. Indeed, we identified multiple alanine substitutions within loop>J of the full length and/or Sec7d of two large brefeldin A-sensitive GEFs (GBF1 and BIG2) and one small brefeldin A-resistant GEF (ARNO) that abrogated binding of ARF and a single alanine substitution that allowed ARF binding but inhibited GDP to GTP exchange. Loop>J sequences are highly conserved, suggesting that loop>J plays a crucial role in the catalytic activity of all ARF GEFs. Using GEF mutants unable to bind ARF, we showed that GEFs associate with membranes independently of ARF and catalyze ARF activation in vivo only when membrane-associated. Our structural, cell biological, and biochemical findings identify loop>J as a key regulatory motif essential for ARF binding and GDP to GTP exchange by GEFs and provide evidence for the requirement of membrane association during GEF activity.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Crystal structure of human BIG2 Sec7d. A, schematic of GBF1, BIG2, and ARNO showing domain arrangement. DCB, dimerization cyclophilin-binding domain; HUS, homology upstream of Sec7d; HDS1–3, homology downstream of Sec7d; CC, coiled coil domain; PH, pleckstrin homology domain. B, alignment of Sec7d from GBF1, BIG2, and ARNO shows sequence conservation in the catalytic F-G loop including the invariant E (boxed in black). The α-helices are marked (A–J). Mutations in loop>J in each Sec7d are boxed and highlighted in red. The loop>J of BIG1 is also shown. Phosphorylation of Ser-883 (boxed and highlighted in blue) inhibits BIG1 exchange activity (8). C, a schematic drawing of the Sec7 domain of human BIG2. The 10 α-helices are labeled A through J. D, overlay of the BIG 2 Sec7 domain (in green) and the complex of Gea1 Sec7 domain (in yellow) and ARF1 (in cyan). GDP and BFA are shown as stick models. Mg2+ is shown as a sphere. Sw. I, switch I region of ARF1; Sw. II, switch II region of ARF1. E, detailed representation of the interactions between the C-terminal loop>J of the Sec7 domain (BIG2 in green and Gea1 in yellow) with the rest of Sec7 and the switch I region of ARF1 (in cyan). The Lys-826 and Ile-827 residues within loop>J make contacts with the H helix and with ARF, respectively.
FIGURE 2.
FIGURE 2.
Expression of GBF1/7A disrupts Golgi morphology. HeLa cells transfected with GFP-GBF1, the catalytically inactive GFP-GBF1/E794K, or the GFP-GBF1/7A mutant were processed for IF using anti-GFP and anti-giantin (gtn) antibodies. Transfected cells are marked with asterisks. Expression of GBF1/E794K and GBF1/7A disrupts Golgi architecture.
FIGURE 3.
FIGURE 3.
Expression of GBF1/7A prevents de novo Golgi biogenesis. HeLa cells were transfected with GFP-GBF1 (A), the catalytically inactive GFP-GBF1/E794K (B), or the GFP-GBF1/7A mutant (C). After 24 h, cells were either mock-treated (− panels), treated with BFA for 1 h (+BFA panels), or treated with BFA followed by a 2-h washout (+BFA +WO panels). Cells were processed for IF using anti-GFP and anti-GM130 antibodies. Insets show enlargements of boxed areas. Wild-type GBF1 supports Golgi reformation, whereas expression of GBF1/E794K and GBF1/7A prevents Golgi biogenesis.
FIGURE 4.
FIGURE 4.
Expression of BIG2/3A causes tubulation of TGN. HeLa cells transfected with HA-tagged wild-type BIG2, the catalytically inactive BIG2/E738K, or the BIG2/3A mutant were processed for IF using anti-HA and anti-γ subunit of the AP1 adaptor complex antibodies. Transfected cells are marked with asterisks. Expression of BIG2/E738K and BIG2/3A causes membrane tubulation.
FIGURE 5.
FIGURE 5.
Mutations in loop>J inhibit GDP to GTP exchange by different mechanisms. GST chimeras of Sec7d from BIG2, BIG2/E738K, and BIG2/3A (A) or from ARNO, ARNO/E156K, and ARNO/1A (B) were purified and tested for nucleotide exchange on myristoylated ARF1 (myrArf1). The data were fit to a single exponential association equation. The data shown are the average of three experiments (error bars, S.E.). Analogous preparations of BIG2 Sec7d (C) or ARNO (D) were immobilized on glutathione beads and incubated with supernatant from cells expressing HA-tagged ARF1/T31N. The postnuclear supernatant (lane PNS) and bound material were analyzed by SDS-PAGE and blotting with anti-HA antibodies. Sec7d from wild-type BIG2 and ARNO show exchange activity, whereas Sec7d from BIG2/E738K, ARNO/E156K, BIG2/3A, and ARNO/1A are catalytically inactive. Sec7d from BIG2/3A does not bind the ARF substrate, whereas ARNO/1A binds ARF but does not catalyze GDP displacement.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jackson C. L., Casanova J. E. (2000) Trends Cell Biol. 10, 60–67 - PubMed
    1. Chardin P., Paris S., Antonny B., Robineau S., Béraud-Dufour S., Jackson C. L., Chabre M. (1996) Nature 384, 481–484 - PubMed
    1. Antonny B., Beraud-Dufour S., Chardin P., Chabre M. (1997) Biochemistry 36, 4675–4684 - PubMed
    1. Mossessova E., Gulbis J. M., Goldberg J. (1998) Cell 92, 415–423 - PubMed
    1. Cherfils J., Ménétrey J., Mathieu M., Le Bras G., Robineau S., Béraud-Dufour S., Antonny B., Chardin P. (1998) Nature 392, 101–105 - PubMed

Publication types

MeSH terms

Associated data

LinkOut - more resources