Structure and Function of Coatomer

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This project addresses the question of intracellular membrane traffic; i.e., how do cells move pieces of membranes from one physical location in the cell to another? While much has been learned about this extremely complicated process over the past twenty years, there are still many questions that remain unanswered. This project focuses on the function of a particular protein complex. COPI, that has been known to be involved in the specific movement of certain membranes from one place to another in the cell and in the specific inclusion of certain membrane proteins in the membranes that are moved.Coat protein I (COPI) is a macromolecular protein complex consisting of ADP-ribosylation factor 1 (ARF1) and coatomer. ARF1 is a small GTP-binding protein that recruits coatomer to Golgi membranes and coatomer is a soluble complex that contains seven protein subunits alpha- through zeta-COP; there's a beta and a beta-prime). After assembly on Golgi membranes, COPI surrounds nascent vesicles that bud from the membrane. Once a vesicle has formed, ARF1 hydrolyzes GTP and COPI dissociates from the membrane, releasing coatomer and ARF1 into the cytosol for another round of coating. Although general features of the COPI cycle are known, the function and molecular mechanism of COPI action are poorly understood. Most researchers agree that COPI-coated vesicles carry membrane in a retrograde direction, from the Golgi to the endoplasmic reticulum (ER), but there is disagreement whether COPI-coated vesicles also carry membrane in the anterograde direction, towards the plasma membrane. It is not understood how COPI binds only to certain membranes, how vesicles coated by COPI form, or precisely how COPI helps select cargo to be included in a budding vesicle. The objective of this project is to better understand the function and action of COPI. Participating in the project will be a post-doctoral fellow, a Ph.D. student, plus rotating graduate and undergraduate students. The work will be done in a university environment where the PI has teaching responsibilities in graduate and undergraduate classes as well as research responsibilities. Coatomer contains sites that bind amino acid motifs present in the cytoplasmic domains of certain transmembrane proteins. Binding of these motifs to coatomer is involved in selecting cargo to be included in a COPI-coated vesicle. The best-characterized cytoplasmic motif is the dilysine signal (KKXX and related sequences) that functions in the return back to the ER of resident ER proteins that have escaped to the Golgi complex. In published work supported by a previous NSF grant, Dr. Draper studied di-amino compounds that appear to mimic the dilysine motif and inhibit the association of beta-COP and delta-COP with Golgi membranes. The research supported by this award is an extension of that previous work and contains two related sub-projects.Sub-project 1 is to investigate changes in coatomer caused by di-amino compounds and peptides. Dr. Draper has recently discovered that certain di-amino compounds that appear to interact with the dilysine binding sites on coatomer dissociate the large coatomer complex into smaller particles. This work will be continued to determine whether coatomer is dissociated into smaller particles by peptides that are natural ligands for coatomer. Coatomer will be incubated with peptides and the formation of sub-complexes will be analyzed by velocity sedimentation centrifugation. Which coatomer subunits are in the sub-complexes will also be determined.Sub-project 2 is to understand the functional consequences of changes in coatomer caused by di-amino compounds and peptides. Dr. Draper has preliminary evidence that sub-complexes formed by coatomer in the presence of di-amino compounds retain partial function, and this work will be extended to study peptides that interact with coatomer. To assess function, Golgi membranes will be incubated with cytosol and an energy source in the presence or absence of peptides. Which coatomer subunits retain Golgi-binding ability will be determined by immunoblotting.This project will thus examine the possible specific role played by the di-lysyl bearing peptides in COPI function. If such a role is substantiated through these and subsequent experiments, it would have a major impact on our understanding of membrane trafficking and specificity. Additionally, the project will serve as a vehicle for the education of the next generation of scientists as well as for the education of scientifically literate members of our society.