Oligomerization of immunoglobulin G heavy and light chains in vitro. A cell-free assay to study the assembly of the endoplasmic reticulum.
A biochemical assay to study the assembly of the endoplasmic reticulum (ER) in a cell-free system is introduced. Incubation in vitro of ER vesicles containing only immunoglobulin gamma 1 heavy (H) chains with ER vesicles containing only K light (L) chains results in fusion and oligomerization of the H and L chains to form the H2L2 complex (immunoglobulin G). ER fusion/H2L2 oligomerization is time and temperature dependent and requires energy in the form of ATP. It is stimulated by the addition of cytosol and requires protease-sensitive components present on the membranes. The addition of guanosine 5'-O-(thiotriphosphate) inhibits membrane fusion and subsequent H2L2 oligomerization without affecting the assembly of H2L2 from detergent-solubilized pools, suggesting an important role for GTPases in vesicle recognition or fusion. The development of a rapid and quantitative assay to study the assembly of the ER in a cell-free system will allow us to identify components involved in the recognition, fusion, and post-fusion events critical for ER function in vivo.
Watkins
J D
JD
Department of Cellular and Molecular Biology, Scripps Research Institute, La Jolla, California 92037.
Hermanowski
A L
AL
Balch
W E
WE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001704:Biopolymers; D002474:Cell-Free System; D002478:Cells, Cultured; D003600:Cytosol; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007147:Immunoglobulin Light Chains / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008561:Membrane Fusion; D051379:Mice
D001704:Biopolymers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D010450:Endopeptidases