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Golgicide A reveals essential roles for GBF1 in Golgi assembly and function

Nature Chemical Biology volume 5, pages 157–165 (2009)Cite this article

Abstract

ADP ribosylation factor 1 (Arf1) plays a critical role in regulating secretory traffic and membrane transport within the Golgi of eukaryotic cells. Arf1 is activated by guanine nucleotide exchange factors (ArfGEFs), which confer spatial and temporal specificity to vesicular transport. We describe here the discovery and characterization of golgicide A, a potent, highly specific, reversible inhibitor of the cis-Golgi ArfGEF GBF1. Inhibition of GBF1 function resulted in rapid dissociation of COPI vesicle coat from Golgi membranes and subsequent disassembly of the Golgi and trans-Golgi network. Secretion of soluble and membrane-associated proteins was arrested at the endoplasmic reticulum–Golgi intermediate compartment, whereas endocytosis and recycling of transferrin were unaffected by GBF1 inhibition. Internalized shiga toxin was arrested within the endocytic compartment and was unable to reach the dispersed trans-Golgi network. Collectively, these results highlight the central role for GBF1 in coordinating bidirectional transport and maintaining structural integrity of the Golgi.

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Figure 1: Discovery of GCA as a potent and effective inhibitor of Stx susceptibility.
Figure 2: GCA disperses medial- and cis-Golgi, inhibits COPI recruitment and maintains localization of AP-1 and GGA3 to TGN.
Figure 3: The effects of GCA are similar to expression of inactive GBF1-E794K.
Figure 4: GCA is selective for GBF1.
Figure 5: GCA causes a decrease in GBF1-dependent Arf1 activation.
Figure 6: GBF1 inhibition arrests secretion of membrane-anchored and soluble proteins.

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Acknowledgements

The authors thank S. Chiang and the Institute of Chemistry and Cellular Biology (ICCB)-Longwood staff for their assistance with screening, J. Loughman (Washington University School of Medicine) for synthesis of MDCK cell cDNA, P. Melançon (University of Alberta) for providing the hamster GBF1 cDNA, M. Vaughn (US National Heart, Lung, and Blood Institute) for providing the BIG1-HA cDNA, M. Haslam for technical assistance and S. Kornfeld and G. Bu for advice and critical review of the manuscript. This work was supported by US National Institutes of Health grant U54 AI057160 to the Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research (MRCE) and by an Investigators in Microbial Pathogenesis Award from the Burroughs Wellcome Foundation.

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  1. José B Sáenz and William J Sun: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, 63110, Missouri, USA

    José B Sáenz, William J Sun, Jinmei Li & David B Haslam

  2. Department of Molecular Microbiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, 63110, Missouri, USA

    José B Sáenz, William J Sun, Jinmei Li & David B Haslam

  3. Department of Cancer Biology, Dana Farber Cancer Institute, 44 Binney Street, Boston, 02115, Massachusetts, USA

    Jae Won Chang & Nathanael S Gray

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 250 Longwood Ave., Boston, 02115, Massachusetts, USA

    Jae Won Chang & Nathanael S Gray

  5. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121, California, USA

    Badry Bursulaya

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Contributions

J.B.S., W.J.S. and J.L. performed experiments. J.B.S. and W.J.S. assisted in manuscript preparation. B.B. performed docking and molecular modeling computations. J.W.C. synthesized and analyzed GCA. N.S.G. designed chemical synthesis, analyzed GCA and assisted in manuscript preparation. D.B.H. designed, performed and analyzed experiments and prepared the manuscript.

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Correspondence to David B Haslam.

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D.B.H. has applied for a patent on the use of Golgicide A as a reagent for cell biology research.

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Sáenz, J., Sun, W., Chang, J. et al. Golgicide A reveals essential roles for GBF1 in Golgi assembly and function. Nat Chem Biol 5, 157–165 (2009). https://doi.org/10.1038/nchembio.144

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