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Vol. 19, Issue 8, 3488-3500, August 2008

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Characterization of Class I and II ADP-Ribosylation Factors (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, John F. Presley, and Paul Melançon^*

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

Submitted April 10, 2008; Revised May 23, 2008; Accepted May 28, 2008
Monitoring Editor: Vivek Malhotra

Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2–5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after 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. In addtion, loss of Arf · GTP after 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).

Abbreviations used: Arf, ADP-ribosylation factor; BFA, brefeldin A; BIGs, BFA inhibited GEFs; COP, coatomer protein; ERES, endoplasmic reticulum exit sites; ERGIC, endoplasmic reticulum-Golgi intermediate compartment; GBF1, Golgi-specific brefeldin A resistance factor 1; GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; IF, immunofluorescence; Man II, mannosidase II; mCherry, monomeric Cherry; NOZ, nocodazole; Sec7d, Sec7 domain.

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