(92f) Evaluating Protein Interactions In the Endoplasmic Reticulum of Saccharomyces Cerevisiae: Spatial Distribution of Molecular Chaperone and Co-Chaperones Evident at a Sub-Organelle Level | AIChE

(92f) Evaluating Protein Interactions In the Endoplasmic Reticulum of Saccharomyces Cerevisiae: Spatial Distribution of Molecular Chaperone and Co-Chaperones Evident at a Sub-Organelle Level


Raden, D. - Presenter, University of Delaware
Robinson, A. S. - Presenter, University of Delaware

The molecular chaperone BiP/Kar2 is a member of the Hsp70 family of chaperones that resides in the endoplasmic reticulum (ER) of S. cerevisiae. BiP is identified in multiple critical processes of the cell including ER translocation, protein folding/maturation, and ER-associated degradation (ERAD). Biochemical and genetic experiments have demonstrated BiP's association with selective co-chaperones for specific ER processes. We hypothesize that chaperone/co-chaperone interactions are a result of spatial inhomogeneity, regulated by co-chaperones, and this inhomogeneity serves as a means of dictating cellular functions. From a systems biology perspective, integration of experimental and computational design will allow us to elucidate the mechanisms associated with chaperone interactions in the ER.

Protein interactions of this system are highly dependent on the timescales of diffusion and kinetics. Diffusion calculations and estimates of species concentrations in vivo must incorporate the physiological conditions of the ER. The low resolution of traditional immunofluorescence techniques combined with BiP's relative high abundance has previously inhibited the determination of protein localization effects at a sub-organelle level as well as the transient behavior associated with co-chaperones. Advances in confocal light microscopy (CLM) combined with the use of green fluorescent protein (GFP) variants allow continuous monitoring of protein dynamics in living cells. Using CLM to visualize fluorescently tagged proteins in vivo, we are able to generate physiologically relevant data of protein dynamics in S. cerevisiae. We have created an in vivo system that investigates the spatial distribution, relative species concentration, and effective diffusion of molecular chaperone BiP and co-chaperones involved in multiple ER processes.

Fusion proteins of BiP and co-chaperone Sec63 have revealed that an inhomogeneous spatial distribution of proteins occurs at a sub-organelle level during translocation. Localization of BiP and Sec63 in the nuclear and peripheral ER has been reconstructed in three dimensions resulting in estimations of ER volume and species concentration. We have confirmed that the ER is continuous and structurally dynamic through Fluorescence Loss in Photobleaching (FLiP) experiments in conjunction with time series analysis of BiP, Sec63, and soluble GFP variants retained in the ER. The dynamics of freely soluble ER resident proteins have been captured at the millisecond timescale. Rapid diffusion of ER resident proteins in combination with an inhomogeneous spatial distribution of chaperone/co-chaperones indicates that we can potentially resolve transient interactions of ER proteins. Our experimental design has directed the development of mathematical models accurately reflecting spatial and temporal effects of chaperone/co-chaperone interactions1,2. The behavior of many systems, including multiple processes of the ER, could not be properly understood without this level of resolution3.

1 Marc Griesemer, Professor Linda Petzold, Department of Computer Science, University of California Santa Barbara

2 Professor Francis Doyle III, Department of Chemical Engineering, University of California Santa Barbara

3 Lemerle, C. et al. Space as the final frontier in stochastic simulations of biological systems. FEBS Letters. (2000) 579:1789-1794.