(820e) 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.