| The translocon-associated protein (TRAP) complex associates with the Sec61 complex and is present in every endoplasmic reticulum (ER) translocon. TRAP has three single membrane-spanning subunits denoted, beta, and delta, while the gamma-subunit spans the membrane four times. TRAP may facilitate protein translocation in the co-translational pathway. However, the mechanism of TRAP function is still not clear.; In this thesis, TRAPalpha and beta lumenal domains (TRAPalpha L and betaL have been co-refolded and co-purified. TRAPalpha LbetaL forms a folded, stable complex based on biochemical and biophysical studies. In particular, CD studies show that TRAPalpha LbetaL is comprised mostly of beta-sheets and turns. Interactions between alphaL-alphaL, alpha L-deltaL, deltaL-deltaL pairs are also observed, although aggregation prevented further investigation. In addition, TRAPalphaLbetaLdeltaL can be co-refolded or co-assembled as a complex, but it is not stable because of a self-aggregation problem with TRAPdeltaL. Our studies suggest that TRAPalpha and beta may have evolved from a common ancestral membrane protein, based on their similar topology, similar predicted secondary structures and phylogenetic sequence analysis. Additional studies on the TRAPalpha LbetaL complex suggest that using shorter constructs, chemical modification or stabilizing additives could lead to crystallization and determination of a structure.; I also purified the native TRAP complex and reconstituted the protein with lipids to form small vesicles and large sheets. In negatively-stained images, the TRAP lumenal domain can be readily observed in edge and front views of vesicles and sheets. Based on these studies, I propose a model of subunit interactions and function within the TRAP complex. TRAPalpha, beta and gamma subunits create the functional core region of TRAP, while TRAPdelta is peripheral to the core region. The lumenal domains of TRAPalpha and beta comprise most of the TRAP lumenal domain, which may directly interact with nascent proteins during translocation or recruit other soluble ER chaperones to the translocon. TRAPgamma may glue together the other three subunits within the ER membrane. Based on recent studies, we propose that TRAP may equilibrate between a monomer and dimer form in the ER membrane, but only a TRAP monomer would interact with the Sec61 complex in the translocon. |
