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MBC in Press, published online ahead of print June 4, 2008
Mol. Biol. Cell 10.1091/mbc.E08-04-0373

A more recent version of this article appeared on August 1, 2008
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Submitted on April 10, 2008
Revised on May 23, 2008
Accepted on May 28, 2008

Characterization of Class I and II Arfs in Live Cells: GDP-bound Class II Arfs Associate with the ER-Golgi Intermediate Compartment Independently of GBF1

Justin Chun,* Zoya Shapovalova,* Selma Y. Dejgaard,{dagger} John F. Presley,{dagger} and Paul Melançon*

*Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada ; {dagger}Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 2B2, Canada

Monitoring Editor: Vivek Malhotra

Despite extensive work on Arf1 at the Golgi complex, the functions of Arf2–5 in the secretory pathway, or for that of any Arf at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1,3,4,5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained GBF1 and the ERGIC marker p58. Unexpectedly, brefeldin A did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but uncovered striking differences between Arf1,3 and Arf4,5. While Arf1,3 quickly dissociated from all endo-membranes following BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. Lastly, loss of Arf•GTP following treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf•GTP, rather than the formation of an Arf•GDP•BFA•GBF1 complex.

Address correspondence to: Paul Melançon (Paul.Melancon{at}UAlberta.ca)




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