Neuroendocrine synaptic vesicles are formed in vitro by both clathrin-dependent and clathrin-independent pathways.
In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.
Shi
G
G
Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.
Faúndez
V
V
Roos
J
J
Dell'Angelica
E C
EC
Kelly
R B
RB
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D008863:Microspheres; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D009490:Neurosecretory Systems / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010750:Phosphoproteins / Q000378:metabolism; D050683:R-SNARE Proteins; D051381:Rats; D013572:Synaptic Vesicles / Q000737:chemistry / Q000378:metabolism*; D016708:Synaptophysin / Q000276:immunology
D033962:Adaptor Protein Complex 2; D033963:Adaptor Protein Complex 3; D033965:Adaptor Protein Complex alpha Subunits; D033942:Adaptor Proteins, Vesicular Transport; D000906:Antibodies; D002352:Carrier Proteins; D002966:Clathrin; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D034041:Monomeric Clathrin Assembly Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D050683:R-SNARE Proteins; D016708:Synaptophysin; C439023:clathrin assembly protein AP180; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors