THE G-PROTEIN-ACTIVATING PEPTIDE, MASTOPARAN, AND THE SYNTHETIC NH2-TERMINAL ARF PEPTIDE, ARFPL3, INHIBIT IN-VITRO GOLGI TRANSPORT BY IRREVERSIBLY DAMAGING MEMBRANES

Citation
Pj. Weidman et Wm. Winter, THE G-PROTEIN-ACTIVATING PEPTIDE, MASTOPARAN, AND THE SYNTHETIC NH2-TERMINAL ARF PEPTIDE, ARFPL3, INHIBIT IN-VITRO GOLGI TRANSPORT BY IRREVERSIBLY DAMAGING MEMBRANES, The Journal of cell biology, 127(6), 1994, pp. 1815-1827
Citations number
54
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Cell Biology
Journal title
ISSN journal
0021-9525
Volume
127
Issue
6
Year of publication
1994
Part
2
Pages
1815 - 1827
Database
ISI
SICI code
0021-9525(1994)127:6<1815:TGPMAT>2.0.ZU;2-6
Abstract
Mastoparan is a cationic amphipathetic peptide that activates trimeric G proteins, and increases binding of the coat protein beta-COP to Gol gi membranes. ARFp13 is a cationic amphipathic peptide that is a putat ive specific inhibitor of ARF function, and inhibits coat protein bind ing to Golgi membranes. Using a combination of high resolution, three- dimensional electron microscopy and cell-free Golgi transport assays, we show that both of these peptides inhibit in vitro Golgi transport, not by interfering in the normal functioning of GTP-binding proteins, but by damaging membranes. Inhibition of transport is correlated with inhibition of nucleotide sugar uptake and protein glycoslation, a decr ease in the fraction of Golgi cisternae exhibiting normal morphology, and a decrease in the density of Golgi-coated buds and vesicles. At pe ptide concentrations near the IC50 for transport, those cisternae with apparently normal morphology had a higher steady state level of coate d buds and vesicles. Kinetic analysis suggests that this increase in d ensity was due to a decrease in the rate of vesicle fission. Pertussis toxin treatment of the membranes appeared to increase the rate of ves icle formation, but did not prevent the membrane damage induced by mas toparan. We conclude that ARFp13 is not a specific inhibitor of ARF fu nction, as originally proposed, and that surface active peptides, such as mastoparan, have the potential for introducing artifacts that comp licate the analysis of trimeric G protein involvement in regulation of Golgi vesicle dynamics.