Intracellular Distribution of Arf Proteins in Mammalian Cells

doi:10.1074/jbc.271.36.21767

Intracellular Distribution of Arf Proteins in Mammalian Cells

Arf6 IS UNIQUELY LOCALIZED TO THE PLASMA MEMBRANE*

From the Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050 and
^§ Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520

** To whom correspondence should be addressed:
Dept. of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd., Atlanta, GA 30322-3050.

↵^¶ Current address: Dept. of Cell Biology, University of Geneva, Sciences III, 30, quai Ernest Ansermet, CH-1211 Geneva 4, Switzerland.
↵^∥ Current address: Dept. of Chemistry, Dickinson College, Carlisle, PA 17013.

Abstract

Subcellular distributions of the five human Arf proteins were examined, using a set of isoform-specific polyclonal and a pan-Arf monoclonal antibodies. Subcellular fractionation of cultured mammalian cells allowed the demonstration that Arf6 is uniquely localized to the plasma membranes of Chinese hamster ovary cells. The plasma membrane distrubution was unaffected by either GTPγS (guanosine 5′-O-(3-thio)triphosphate) or brefeldin A, an activator and inhibitor of Arf activities, respectively. In contrast, Arf proteins 1, 3, 4, and 5 were predominantly cytosolic but could be recruited to a variety of intracellular membranes, but not plasma membranes, upon incubation in the presence of GTPγS. The GTPγS-promoted binding of the cytosolic Arf proteins to membranes was blocked by brefeldin A. The stable association of Arf6 with plasma membranes and the insensitivity of its localization to either GTPγS or brefeldin A revealed a clear distinction between Arf6 and the other Arf isoforms. Localization of Arf6 to the plasma membrane suggests a unique cellular role for this isoform at the plasma membrane, but failure to find endogenous Arf6 on endocytic structures, including clathrin-coated vesicles, appears inconsistent with the proposed role of Arf6 in assembly of coat structures or endosomes in transfected fibroblasts (1, 2).

Footnotes

↵^‡ The first two and last two authors contributed equally to this work.
↵* This work was supported in part by grants from the National Institutes of Health and Human Frontier Science Program (to I. M.) and by the intramural program of the Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute (to R. A. K.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

Arf

ADP-ribosylation factor

Arl

Arf-like

BFA

brefeldin A

CHO

Chinese hamster ovary cells

ELISA

enzyme-linked immunosorbent assay

ER

endoplasmic reticulum

ERGIC

ER/Golgi intermediate compartment

FFE

free flow electrophoresis

GTPγS

guanosine 5′-O-(3-thio)triphosphate

HRP

horseradish peroxidase.
↵²C. J. Zhang, K. Johnson, M. C. Willingham, W. B. Greene, A. G. Rosenwald, and R. A. Kahn, submitted for publication.
↵³M. M. Cavenagh, R. Kamath, and R. A. Kahn, manuscript in preparation.
↵⁴Boman, A. L., Taylor, T. C., Berger, S. J., Melancon, P., and Wilson, K. L. (1996) Biochemistry 35, 8244-8251
↵⁵J. Donaldson, personal communication.
↵⁶M. M. Cavenagh, J. A. Whitney, K. Carroll, C. Zhang, A. L. Boman, A. G. Rosenwald, I. Mellman, and R. A. Kahn, unpublished data.
- Received February 26, 1996.
- Revision received June 10, 1996.