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Kimberly Cope

Advisor: Kevin. A. Morano, Ph.D.

The majority of proteins require molecular chaperones to assist their folding into tertiary and quaternary structures. Certain stresses can compromise the weak hydrophobic forces responsible for these structures and lead to protein unfolding, misfolding, and aggregation. Aggregates of proteins are hallmarks of devastating diseases such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. Fortunately, bacteria, plants, and fungi have a potent disaggregase, named Hsp104 in Saccharomyces cerevisiae.

Recently, heat-induced aggregates, termed Q-bodies, were found to contain three molecular chaperones: Hsp70, Hsp104, and Hsp42. Their coalescence from small puncta into larger inclusions required Hsp104. During glucose deprivation, a stress that isn’t known to cause unfolding of proteins, I have observed Hsp104, Hsp26, and Hsp42 but not Ssa1 form into cytoplasmic foci. As the Q-bodies are hypothesized to be involved in protein refolding or degradation, the starvation-induced bodies may also play a crucial role in protein quality control. The overall goal of my project was to characterize these novel bodies in terms of composition and dynamics. The different chaperones coalesce into aggregates at different frequencies in response to starvation, however a thermally labile protein (firefly luciferase) does not, suggesting that misfolded proteins are not responsible for the recruitment of the chaperones. One hypothesis is that energy depletion is hampering the ability of certain proteins to be anchored into the membrane posttranslationally, exposing hydrophobic residues prone to aggregation. By performing the glucose deprivation in the presence of cycloheximide, a translation inhibitor, I observed that, indeed, newly synthesized proteins must be present for the formation of CBs.

To determine whether CBs colocalize and share a purpose with other known foci, I compared the localization of the chaperones to representative markers in live cells. After acute glucose starvation, Hsp104 partially colocalizes with proteasome storage granules and fully overlaps with stationary phase bodies. I observed that Hsp104’s localization patterns were partially dependent on the nature of the fluorescent protein attached. Fusion to yEmRFP caused Hsp104 to remain mostly diffuse during starvation, while fusion to CFP caused it to localize to structures that resemble the septin ring and Gln1 filaments. CFP also caused an apparent cytokinesis defect.

Research Info

Energy stress causes chaperones to assemble into cytoplasmic complexes