Binding and Functions of ADP-ribosylation Factor on Mammalian and Yeast Peroxisomes

Hans Heid,Bianka L. Grosshans,Wilhelm W. Just,Dorothee Lay,Karin Gorgas

doi:10.1074/jbc.m503497200

Abstract

We have analyzed in vitro the binding characteristics of members of the ADP-ribosylation factor (ARF) family of proteins to a highly purified rat liver peroxisome preparation void of Golgi membranes and studied in vivo a role these proteins play in the proliferation of yeast peroxisomes. Although both ARF1 and ARF6 were found on peroxisomes, coatomer recruitment only depended on ARF1-GTP. Recruitment of ARF1 and coatomer to peroxisomes was significantly affected both by pretreating the animals with peroxisome proliferators and by ATP and a cytosolic fraction designated the intermediate pool fraction depleted of ARF and coatomer. In the presence of ATP, the concentrations of ARF1 and coatomer on peroxisomes were reduced, whereas intermediate pool fraction led to a concentration-dependent decrease in ARF and increase in coatomer. Brefeldin A, a fungal toxin that is known to reduce ARF1 binding to Golgi membranes, did not affect ARF1 binding to peroxisomes. In Saccharomyces cerevisiae, both ScARF1 and ScARF3, the yeast orthologs of mammalian ARF1 and ARF6, were implicated in the control of peroxisome proliferation. ScARF1 regulated this process in a positive manner, and ScARF3 regulated it in a negative manner.

Highlights

The intracellular communication of peroxisomes with other subcellular compartments is only poorly explored
Our results demonstrate that (i) ADP-ribosylation factor (ARF)-GTP binding to peroxisomes in vitro is essential for subsequent binding of coatomer, (ii) it is the subtype 1 of ARF that favors coatomer recruitment, (iii) ARF and coatomer binding is affected by ATP and a cytosolic factor but is not inhibited by brefeldin A (BFA), and (iv) besides ARF1, ARF6 is found on peroxisomal membranes
The results suggest that ARF1 is essential for coatomer recruitment to peroxisomes and that, besides ARF1, ARF6 may be involved in the control of peroxisomal processes

Summary

EXPERIMENTAL PROCEDURES

Materials—AMP-PNP, GMP-PNP, and GDP␤S were purchased from Sigma, and ATP and creatine kinase were from Roche Applied Science. To purify the expressed proteins, cells were harvested, resuspended in lysis buffer (50 mM Tris/HCl, pH 8.0, 10 mM imidazol, 5 mM ␤-mercaptoethanol, 200 ␮M GDP, 1 ␮M MgCl2) supplemented with the protease inhibitor mixture Complete (Roche Applied Science), cleared for 5 min at 500 ϫ g, 4 °C, and lysed using an EmulsiFlex-C5 (Avestin Inc., Mannheim, Germany) according to the manufacturer’s protocol. Recombinant ARF eluting at 170 –190 mM imidazol was pooled, concentrated 2–3-fold (Vivaspin 20/molecular weight cut-off 5,000; Vivascience, Hannover, Germany), dialyzed against 20 mM Tris/HCl, pH 7.4, 1 mM MgCl2, 1 mM dithiothreitol, 5 ␮M GDP, and stored in aliquots at Ϫ80 °C. Mass Spectrometry and Edman Sequencing—Peroxisomal membranes were prepared by carbonate treatment [28] and incubated with APF and GMP-PNP as described above for intact peroxisomes. Samples were treated with 1 M KCl for 30 min on ice, and the membranes were

TABLE ONE

RESULTS

Corresponding peptides

DISCUSSION