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Molecular chaperones play a central role in maintaining protein homeostasis. The highly conserved Hsp70 family of chaperones have major functions in folding of nascent peptides, protein refolding, and protein aggregate disassembly. In yeast, loss of two Hsp70 proteins, Ssa1 and Ssa2, is associated with decreased cellular growth and shortened lifespan. While heterologous or mutant temperature-sensitive proteins form anomalous large cytoplasmic inclusions in ssa1Δssa2Δ strains, it is unclear how endogenous wild-type proteins behave and are regulated in the presence of limiting Hsp70s. Using the wild-type yeast Poly A binding protein (Pab1), which is involved in mRNA binding and forms stress granules (SGs) upon heat shock, Pab1 forms large inclusions in approximately half of ssa1Δssa2Δ cells in the absence of stress. Overexpression of Ssa1, Hsp104, and Sis1 almost completely limits the formation of these large inclusions in ssa1Δssa2Δ, suggesting that excess Ssa1, Hsp104, and Sis1 can each compensate for the lower levels of Ssa proteins. Upon heat shock, SGs also form in cells whether large Pab1 inclusions are present or not. Surprisingly, cells containing only SGs disassemble faster than wild type, whereas cells with both large inclusions disassemble slower albeit completely. We suspect that disassembly of these large inclusions is linked to the elevated heat shock response and elevated Hsp104 and Sis1 levels in ssa1Δssa2Δ strains. We also observed that wild-type cultures grown to saturation also form large Pab1-GFP inclusions. These inclusions can be partially rescued by overexpression of Ssa1. Taken together, our data suggest that Hsp70 not only plays a role in limiting unwanted protein aggregation in normal cells, but as cells age, the depletion of active Hsp70 possibly underlies the age-related aggregation of endogenous proteins. 

Molecular chaperones play a central role in protein disaggregation. However, the molecular determinants that regulate this process are poorly understood. Hsp104 is an AAA+ ATPase that disassembles stress granules and amyloids in yeast through collaboration with Hsp70 and Hsp40.In vitrostudies show that Hsp104 processes different types of protein aggregates by partially translocating or threading polypeptides through the central pore of the hexamer. However, it is unclear how Hsp104 processing influences client protein functionin vivo. The middle domain (MD) of Hsp104 regulates ATPase activity and interactions with Hsp70. Here, we tested how MD variants, Hsp104^A503Sand Hsp104^A503V, process different protein aggregates. We establish that engineered MD variants fail to resolve stress granules but retain prion fragmentation activity required for prion propagation. Using the Sup35 prion protein, ourin vitroandin vivodata indicate that the MD variants can disassemble Sup35 aggregates, but the disaggregated protein has reduced GTPase and translation termination activity. These results suggest that the middle domain can play a role in sensing certain substrates and plays an essential role in ensuring the processed protein is functional. 

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin’s precise role is unclear. Here, we investigate how actin influences the cell’s ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.