Abstract
The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.
Highlights
When we transfected HA-tagged GLUT1 into PC12 cells and examined its distribution relative to synaptic vesicle proteins in glycerol velocity gradients, we found that the majority of the protein was found in heavier membranes, including the plasma membrane and endosomes as described previously [23], a peak of GLUT1HA immunoreactivity was reproducibly localized to a region of the gradient that overlapped with but was clearly distinct from the peak of immunoreactivity for the synaptic vesicle protein synaptophysin (Fig. 1A)
We have identified a vesicular compartment in PC12 cells with a counterpart in rat brain that is characterized by the colocalization of synaptic vesicle proteins with a variety of other proteins, including multiple different transporters and the transferrin receptor
In addition to providing evidence for cosedimentation of membranes containing these proteins with synaptic vesicle proteins, we show by organelle immunoisolation that synaptic vesicle proteins and GLUT1 are in the same vesicle and that these vesicles are not derived from a shared component of the biosynthetic pathway or by vesiculation of the plasma membrane
Summary
Cell Culture and Transfection—PC12 cells were grown in high glucose Dulbecco’s modified Eagle’s media (Life Technologies, Inc.) supplemented with 2 mM glutamine, 10 units/ml penicillin/streptomycin, 5% horse serum, and 5% defined/supplemented bovine calf serum (HyClone, Logan, UT) in a humidified 37 °C incubator at 10% CO2. The samples were incubated on ice for 10 min, spun for 30 min at 13,000 rpm in a microcentrifuge at 4 °C, and the pellets were resuspended in 20 l of 1ϫ SDS sample buffer consisting of 8 M urea, 5% SDS, 20% glycerol, 1 mM EDTA, 1 M 2-mercaptoethanol, and 0.25 M Tris, pH 6.8. 0.6 mg of goat anti-mouse IgG-coated M-500 Dynabeads (Dynal, Great Neck, NY) were coated with 0.5 g of mAb SY38 This amount of beads plus primary antibody was incubated with one gradient fraction overnight at 4 °C. The resulting pellet (P2) was resuspended in 5 ml of ice-cold homogenization buffer, layered on top of 20 ml of 0.8 M sucrose in 5 mM HEPES, 0.1 mM EGTA, pH 7.4, and spun in an SS34 rotor at 8500 rpm for 25 min at 4 °C. Sample volumes were adjusted to 30 l with 1% Nonidet P-40, enzyme digestion buffer, and 250 units of endoglycosidase H (Endo H; New England Biolabs, Beverly, MA) or 250 units of peptide:N-glycosidase F (PNGase F; New England Biolabs) and incubated 16 h at RT
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