Oligomerization of immunoglobulin G heavy and light chains in vitro. A cell-free assay to study the assembly of the endoplasmic reticulum.

Abstract

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.