An AP-3-dependent mechanism drives synaptic-like microvesicle biogenesis in pancreatic islet beta-cells.
Pancreatic islet beta-cells contain synaptic-like microvesicles (SLMVs). The origin, trafficking, and role of these SLMVs are poorly understood. In neurons, synaptic vesicle (SV) biogenesis is mediated by two different cytosolic adaptor protein complexes, a ubiquitous AP-2 complex and the neuron-specific AP-3B complex. Mice lacking AP-3B subunits exhibit impaired GABAergic (inhibitory) neurotransmission and reduced neuronal vesicular GABA transporter (VGAT) content. Since beta-cell maturation and exocytotic function seem to parallel that of the inhibitory synapse, we predicted that AP-3B-associated vesicles would be present in beta-cells. Here, we test the hypothesis that AP-3B is expressed in islets and mediates beta-cell SLMV biogenesis. A secondary aim was to test whether the sedimentation properties of INS-1 beta-cell microvesicles are identical to those of bona fide SLMVs isolated from PC12 cells. Our results show that the two neuron-specific AP-3 subunits beta3B and mu3B are expressed in beta-cells, the first time these proteins have been found to be expressed outside the nervous system. We found that beta-cell SLMVs share the same sedimentation properties as PC12 SLMVs and contain SV proteins that sort specifically to AP-3B-associated vesicles in the brain. Brefeldin A, a drug that interferes with AP-3-mediated SV biogenesis, inhibits the delivery of AP-3 cargoes to beta-cell SLMVs. Consistent with a role for AP-3 in the biogenesis of GABAergic SLMV in beta-cells, INS-1 cell VGAT content decreases upon inhibition of AP-3 delta-subunit expression. Our findings suggest that beta-cells and neurons share molecules and mechanisms important for mediating the neuron-specific membrane trafficking pathways that underlie synaptic vesicle formation.
Suckow
Arthur T
AT
1Department of Medicine and Pediatric Diabetes Research Center, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0983, USA.
Craige
Branch
B
Faundez
Victor
V
Cain
William J
WJ
Chessler
Steven D
SD
D000818:Animals; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D007328:Insulin / Q000502:physiology; D050417:Insulin-Secreting Cells / Q000502:physiology*; D018613:Microscopy, Confocal; D016716:PC12 Cells; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013572:Synaptic Vesicles / Q000502:physiology*; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D050495:Vesicular Inhibitory Amino Acid Transport Proteins / Q000502:physiology
D004268:DNA-Binding Proteins; D007328:Insulin; D011500:Protein Synthesis Inhibitors; C118623:Slc32a1 protein, rat; D014157:Transcription Factors; D050495:Vesicular Inhibitory Amino Acid Transport Proteins; C060759:enhancer-binding protein AP-3; D020126:Brefeldin A; D012313:RNA