A complete understanding of the complexities of cellular proliferation and neoplasia will require a thorough analysis of the biochemical and molecular events governing the structure and function of organelles of the secretory pathway in eukaryotic cells. The long range objective of our research is to understand the structural and functional organization of the endoplasmic reticulum (ER). The research supported by this grant presents a new quantitative assay we have developed to study the molecular mechanisms governing ER structure in a cell-free system. This new assay is based on the concept that one of the major functions of the ER is to catalyze the folding and assembly of homo- and heterooligomeric proteins. Using interphase ER prepared from two different hybridoma cell lines which express either the heavy (H) or light (L) chains of the mature IgG molecule, we have established in vitro conditions which support the fusion and mixing of the lumenal contents of the two ER compartments leading to the oligomerization of the H and L subunits into the mature IgG molecule (H2L2).
Our specific aims are to thoroughly characterize the basic biochemical requirements for ER fusion and IgG oligomerization to define the soluble and membrane-associated components which regulate the structure of interphase ER.These studies will include purification of highly enriched ER fractions, optimization of assay conditions which promote ER fusion, biochemical and kinetic analysis of H and L chain oligomerization into mature IgG, characterization of energy requirements, and analysis of specificity of fusion. The soluble cytosolic and membrane-associated proteins promoting ER fusion will be characterized by biochemical fractionation of the key components, their molecular cloning, and preparation of both monoclonal and polyclonal antibody reagents to study the function of these proteins in vitro. NSF, NSF receptor and SNAPs, proteins involved in membrane fusion in vesicular trafficking, will be examined for their potential role in ER assembly. In addition, members of the rab gene family of small GTP-binding proteins will be evaluated for their potential role in regulation of ER structure. To understand the differences in the biochemical mechanisms regulating ER structure during the cell, cycle, we will examine the hypothesis that a kinase/phosphatase cascade initiated by the p34cdc2 kinase is responsible for the phosphorylation and dephosphorylation of key components involved in the disassembly and assembly of the ER during entry and exit from mitosis. Specifically, we will prepare highly enriched metaphase ER and cytosol, and purified p34cdc2 kinase to study cell cycle related modifications leading to the disassembly or assembly of the ER. These studies will be pursued in conjunction with experiments which explore the ability of the phosphatase inhibitor okadaic acid (OKA) to inhibit ER assembly in vitro, potentially revealing a cell cycle specific kinase controlling ER structure.
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