TY - JOUR
T1 - Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes
AU - Clift-O'Grady, Lois
AU - Desnos, Claire
AU - Lichtenstein, Yael
AU - Faúndez, Victor
AU - Horng, Jim Tong
AU - Kelly, Regis B.
PY - 1998/10
Y1 - 1998/10
N2 - Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15°C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15°C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15°C, vesicles generated in vitro at 37°C had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.
AB - Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15°C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15°C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15°C, vesicles generated in vitro at 37°C had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system.
UR - http://www.scopus.com/inward/record.url?scp=0031782966&partnerID=8YFLogxK
U2 - 10.1006/meth.1998.0662
DO - 10.1006/meth.1998.0662
M3 - 文章
C2 - 9790861
AN - SCOPUS:0031782966
SN - 1046-2023
VL - 16
SP - 150
EP - 159
JO - Methods: A Companion to Methods in Enzymology
JF - Methods: A Companion to Methods in Enzymology
IS - 2
ER -