Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes.
Year: 1998
Type: article
Abstract: We have recently become aware that the cDNA for human ARF5 that we used in our study encoded amino acids 3–17 of cAMP-dependent protein kinase instead of amino acids 3–17 of mARF5. Because this finding raised questions about the validity of the results obtained with this chimeric ARF5, we obtained an authentic cDNA for human ARF5 from Dr. Joel Moss (National Institutes of Health), prepared myristoylated mARF5 as described in our paper, and repeated the experiments. As shown in Table I below, the overall Golgi-stimulated guanine nucleotide exchange for myristoylated mARF5 was greater than that observed with myristoylated mARF1. The brefeldin A (BFA) inhibitable component, which is a measure of the exchange catalyzed by the Golgi guanine nucleotide exchange factor (GEF), was less for mARF5 than for mARF1 (18.4 pmol/nmol myr mARF5 versus 25.6 pmol/nmol myr mARF1). This indicates that authentic myristoylated mARF5 interacts with the Golgi GEF about 70% as well as myristoylated mARF1. The BFA-resistant exchange is presumably due to interactions with the Golgi membrane phospholipids. The activation of mARF5 by the Golgi GEF is considerably better than that reported for the chimeric mARF5 (11% of the mARF1 value). The mARF5 promoted AP-1 coat protein binding to the Golgi membranes to the same extent as myristoylated mARF1, consistent with the data reported for the chimeric mARF5.Table IGolgi-stimulated guanine nucleotide exchange on ARF1 and ARF5GTPγS boundGolgi-stimulated exchangepmolpmolpmol/nmol myr ARFmyr mARF13.40 ± 0.46Golgi0.84 ± 0.05myr mARF1 + Golgi11.68 ± 1.087.429.8myr mARF1 + Golgi + BFA5.28 ± 0.421.04.2myr mARF56.38 ± 0.88Golgi0.84 ± 0.05myr mARF5 + Golgi22.50 ± 0.9215.361.1myr mARF5 + Golgi + BFA17.90 ± 1.1210.742.7 Open table in a new tab We conclude from these experiments that myristoylated mARF5 interacts with the Golgi GEF almost as well as myristoylated ARF1 and that once bound to the Golgi membranes, both ARFs facilitate AP-1 recruitment equally. We thank Dr. Romero (University of Pittsburgh) for calling our attention to the problem with the mARF5 cDNA and Dr. Joel Moss for generously supplying the human ARF5 cDNA and highly purified myristoylated mARF5.
Source: Journal of Biological Chemistry
Authors: Jennifer Ostrom Liang, Stuart Kornfeld
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W4239557491