(34f) Evaluating Organelle Dynamics & Protein Interactions in the Endoplasmic Reticulum of Saccharomyces Cerevisiae
The first organelle of the secretory pathway, the endoplasmic reticulum (ER), is responsible for multiple intracellular processes including ER protein translocation, protein folding/maturation, karyogamy, and ER-associated degradation (ERAD). BiP/Kar2 is the molecular chaperone that resides in the ER lumen of S. cerevisiae. Biochemical and genetic experiments have demonstrated BiP's association with selective co-chaperones in all aforementioned processes. We hypothesize that compartmentalization of ER resident proteins occurs at the sub-organelle level and is the basis of ER protein interactions and organelle biogenesis. Furthermore, we believe that the spatial heterogeneity of BiP is regulated by co-chaperones, and this heterogeneity serves as a means of dictating cellular functions. To generate physiologically-relevant data of protein dynamics, variants of green fluorescent protein (GFP) coupled with advances in confocal light microscopy techniques have allowed us to track multiple fluorescently-tagged proteins in vivo.
Dual expression strains composed of fusion proteins, BiP and co-chaperones Sec63 or Scj1, reveal that a heterogeneous spatial distribution is evident at the sub-organelle level. Secondary confirmation of our results has been performed using immunofluorescence techniques in multiple S. cerevisiae strains. We have captured the spatiotemporal effects of protein dynamics in live cells by monitoring ER proteins involved in translocation (Sec61, Sec63), protein folding (Scj1, PDI), and ERAD (Doa10, Hrd1, Sec61); monitored rapid diffusion and trafficking of ER resident proteins; as well as ER morphology under various environmental conditions. We have confirmed that the ER is continuous through Fluorescence Loss in Photobleaching (FLiP) experiments and evaluated the colocalization of ER resident proteins compared to the cytoskeleton in order to elucidate the mechanisms associated with ER biogenesis. Integration of our experimental data with computational design will enable us to develop stochastic models of biological systems that accurately reflect spatiotemporal effects of ER protein interactions.