The G protein-activating peptide, mastoparan, and the synthetic NH2-terminal ARF peptide, ARFp13, inhibit in vitro Golgi transport by irreversibly damaging membranes.
Mastoparan is a cationic amphipathetic peptide that activates trimeric G proteins, and increases binding of the coat protein beta-COP to Golgi membranes. ARFp13 is a cationic amphipathic peptide that is a putative specific inhibitor of ARF function, and inhibits coat protein binding 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 decrease in the fraction of Golgi cisternae exhibiting normal morphology, and a decrease in the density of Golgi-coated buds and vesicles. At peptide concentrations near the IC50 for transport, those cisternae with apparently normal morphology had a higher steady state level of coated buds and vesicles. Kinetic analysis suggests that this increase in density was due to a decrease in the rate of vesicle fission. Pertussis toxin treatment of the membranes appeared to increase the rate of vesicle formation, but did not prevent the membrane damage induced by mastoparan. We conclude that ARFp13 is not a specific inhibitor of ARF function, as originally proposed, and that surface active peptides, such as mastoparan, have the potential for introducing artifacts that complicate the analysis of trimeric G protein involvement in regulation of Golgi vesicle dynamics.
Weidman
P J
PJ
Department of Biochemistry and Molecular Biology, St. Louis University Medical School, Missouri 63104.
Winter
W M
WM
D020727:ADP-Ribosylation Factors; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D003600:Cytosol / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006056:Golgi Apparatus / Q000187:drug effects* / Q000648:ultrastructure; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D008566:Membranes / Q000502:physiology; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology*; D010455:Peptides; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000378:metabolism; D014862:Wasp Venoms / Q000494:pharmacology*
C028391:G protein, vesicular stomatitis virus; D036341:Intercellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D009711:Nucleotides; D010446:Peptide Fragments; D010455:Peptides; D014759:Viral Envelope Proteins; D014862:Wasp Venoms; C037500:mastoparan; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors