The Exploitation of Yeast – Elucidating Quality Control & Regulation in the Secretory Pathway

Within the secretory pathway of eukaryotic cells, the endoplasmic reticulum (ER) is responsible for maintaining the fidelity of protein synthesis and maturation. A variety of disturbances including nutrient deprivation, pathogenic infection, and chemical treatment, collectively termed 'ER stress', induce quality control mechanisms to facilitate the recovery of cell homeostasis. ER-associated degradation (ERAD), unfolded protein response (UPR), and autophagy are quality control pathways that occur on various timescales, encompass variations in the spatial organization of multiple organelles, and alter select protein concentrations and intracellular localization. Surprisingly, all three pathways are activated in several neurodegenerative and hereditary diseases. In all cases, as a result of ER stress, there is evidence of atypical, intracellular protein distribution during disease manifestation. Yet, how the accumulation of disease-specific proteins is involved in compromising quality control remains elusive.

By implementing DNA recombination strategies combined with high-resolution imaging techniques, we determined that protein redistribution, resultant spatial changes, and organelle modifications are a consequence of the cell's response to ER stress in the yeast, S. cerevisiae. In pursuit of a thorough analysis of the protein redistribution at the subcellular level, multiple yeast expression cassettes have been created to test the effects of codon-optimized fluorescent protein (FP) variants [1], small epitope tags (reviewed in [2]), polylinker length for N- and C-terminal tags, and the inclusion of essential retrieval sequences for ER resident proteins [3]. Consequently, our approach enables one to monitor the trafficking effects of ER chaperones and foldases in real time by incorporating H/KDEL retrieval sequences fused to FP variants. To investigate discrete subpopulations of tagged proteins, live-cell imaging methods and super-resolution techniques (e.g. Fluorescence-Photoactivation Localization Microscopy, F-PALM and Structured Illumination Microscopy, SIM) were used to evaluate organelle morphology. Focused-Ion Beam Scanning Electron Microscopy (FIB-SEM) achieved electron microscope resolution, which facilitated entire three-dimensional organelle reconstructions of yeast cells [6]. Interestingly, the improved spatial resolution confirmed organelle connectivity during periods of prolonged stress (e.g. peripheral ER and mitochondria junctions) and inheritance (e.g. lipid droplets derived from perinuclear ER).

Utilizing FP variants as probes, proteins were recombinantly expressed from their native promoter in order to monitor protein trafficking, analyze localization effects of proteins involved in ERAD, and examine organelle dynamics and morphology as a consequence of local perturbations to the system [4]. Consequently, we confirmed the existence of cellular variability following UPR activation and established that endogenous proteins redistribute in the cell – specifically the ER – in order to perform essential functions that maintain cell homeostasis. To establish that the UPR is a global response to localized perturbations, we evaluated the transcriptional effects of UPR activation. As a result, microarray analysis and q-PCR validation have confirmed the novel repression of 206 genes – highly enriched in protein synthesis and metabolic biological functions – following ER stress [5]. Our results expand the characterization of the UPR and reaffirm diverse, global consequences for the cell.

Due to the intrinsic complexity of biological systems, the integration of a broad spectrum of molecular engineering techniques and assays combined with novel imaging analyses is crucial when investigating regulation of the secretory pathway. Collectively, our experimental approaches have led to a better understanding of ER homeostasis and cell regulation, thus facilitating the implementation of new therapeutic modalities aimed at stress reduction.

References

1.     C. L. Young, D. Raden, J. Caplan, K. Czymmek, A. S. Robinson Optimized Cassettes for Live-Cell Imaging of Proteins and High Resolution Techniques in Yeast, Yeast, 2012 doi:10.1002/yea.2895. [Epub 2012 Apr 4]

2.     C. L. Young, Z. T. Britton, A. S. Robinson Recombinant Protein Expression and Purification: A Comprehensive Review of Affinity Tags and Microbial Applications, Biotechnology Journal, 7(4), Jan 10 2012 doi:10.1002/biot.201100155. [Epub ahead of print]

3.     C. L. Young, D. L. Raden, A. S. Robinson, Analysis of Endoplasmic Reticulum Resident Proteins in S. cerevisiae: Implementation of H/KDEL Retrieval Sequences, Traffic, 2013 apr;14(4):365-81. doi:10.111/tra.12041. Epub 2013 Feb 4.

4.     C. L. Young, D. L. Raden, J. Caplan, A. S. Robinson Dynamics of Endoplasmic Reticulum Resident Proteins and Organelle Morphology in S. cerevisiae, 2013 (in preparation).

5.     D. Wei, S. Jacobs, S. Modla, S. Zhang, C. L. Young, R. Cirino, J. Caplan, K. Czymmek High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy, BioTechniques, 2012 Jul;53(1):41-8.

6.     T. Yuraszeck , C. Young , P. Xu, C. A. Gelmi, F. J. Doyle III, A. S. Robinson Novel down-regulation pathways in the Unfolded Protein Response from S. cerevisiae provide evidence of a complex regulatory response to ER stress, 2012 (under revision)  co-authorship.