Expression of a dominant allele of human ARF1 inhibits membrane traffic in vivo

• Type of Material: Article
• Authors: Chun-jiang Zhang, National Institutes of Health; Anne G. Rosenwald, National Institutes of Health; Mark C. Willingham, National Institutes of Health; Susan Skuntz, National Institutes of Health; Jenny Clark, National Institutes of Health; Richard A Kahn, Emory University
• Language: English
• Date: 1994-02-01
• Publisher: Rockefeller University Press
• Publication Version: Final Published Version
• Copyright Statement: © The Rockefeller University Press
• License: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1083/jcb.124.3.289
• Title of Journal or Parent Work: Journal of Cell Biology
• ISSN: 0021-9525
• Volume: 124
• Issue: 3
• Start Page: 289
• End Page: 300
• Keywords: ORGANELLE STRUCTURE; GS-ALPHA; Science & Technology; BETA-COP; BREFELDIN-A; ADP-RIBOSYLATION FACTOR; GTP-BINDING-PROTEIN; COATED VESICLES; ESCHERICHIA-COLI; GUANINE-NUCLEOTIDE; Cell Biology; Life Sciences & Biomedicine; GOLGI MEMBRANES
• Research Categories: Chemistry, Biochemistry; Health Sciences, Pathology

Abstract

ADP-ribosylation factor (ARF) proteins and inhibitory peptides derived from ARFs have demonstrated activities in a number of in vitro assays that measure ER-to-Golgi and intra-Golgi transport and endosome fusion. To better understand the roles of ARF proteins in vivo, stable cell lines were obtained from normal rat kidney (NRK) cells transfected with either wild-type or a dominant activating allele ([Q71L]) of the human ARF1 gene under the control of the interferon-inducible mouse Mx1 promoter. Upon addition of interferon, expression of ARF1 proteins increased with a half-time of 7-8 h, as determined by immunoblot analysis. Induction of mutant ARF1, but not wild-type ARF1, led to an inhibition of protein secretion with kinetics similar to that observed for induction of protein expression. Examination of the Golgi apparatus and the ER by indirect immunofluorescence or transmission electron microscopy revealed that expression of low levels of mutant ARF1 protein correlated with a dramatic increase in vesiculation of the Golgi apparatus and expansion of the ER lumen, while expression of substantially higher levels of wild-type ARF1 had no discernible effect. Endocytosis was also inhibited by expression of mutant ARF1, but not by the wild-type protein. Finally, the expression of [Q71L]ARF1, but not wild-type ARF1, antagonized the actions of brefeldin A, as determined by the delayed loss of ARF and beta-COP from Golgi membranes and disruption of the Golgi apparatus. General models for the actions of ARF1 in membrane traffic events are discussed.