Plasma membrane resident 'fusion complexes' mediate reconstituted exocytosis.
Calcium-triggered exocytosis is thought to be mediated by membrane-associated protein complexes. In sea urchin eggs, high concentrations of calcium activate multiple 'fusion complexes' per cortical vesicle-plasma membrane docking site. Some of these fusion complexes are known to reside in the vesicle membrane. It is not known if fusion complexes also reside in the plasma membrane, or if plasma membrane-resident fusion complexes require cognate partners in the vesicle membrane. Using reconstitution, we show that N-ethylmaleimide treatment of either vesicles or plasma membrane fragments prior to reconstitution does not completely inhibit exocytosis. Treatment of both components did result in complete inhibition. Upon reconstitution, cortical vesicles and the early endosomes formed by compensatory endocytosis both contributed, on average, two fusion complexes per reconstituted docking site. The plasma membrane contributed, on average, two fusion complexes per docking site when assembled with cortical vesicles, but only one complex when reconstituted with endosomes. We conclude that there are at least two types of plasma membrane-resident fusion complexes that participate in reconstituted cortical vesicle-plasma membrane fusion. The activity of one of these fusion complexes is target-specific for cortical vesicles, while the second type also supports fusion with endosomes.
Ikebuchi
Y
Y
Institute of Molecular Medicine and Genetics, Department of Medicine, Medical College of Georgia, 1120 15th Street CB 2803. Augusta, GA 30912-2630, USA.
Baibakov
B
B
Smith
R M
RM
Vogel
S S
SS
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D015638:Cytochalasin D / Q000494:pharmacology; D011992:Endosomes / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012617:Sea Urchins / Q000378:metabolism; D013997:Time Factors
D000199:Actins; D019384:Nucleic Acid Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D015638:Cytochalasin D; D005033:Ethylmaleimide; D002118:Calcium