(Georgia Institute of Technology, 2022-07-25)
Jay Garcia, Lina Manuela
Misfolding occurs when proteins fail to fold into their proper functional state. Normally, most misfolded proteins refold or are targeted for degradation through a special network of chaperones in order to maintain cellular proteostasis. However, in some cases, misfolded proteins can form ordered fibrous aggregates called amyloids. Amyloids are generally associated with aging, with neurological disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, and with a variety of other disorders, such as Type II diabetes and atherosclerosis.
Yeast transmissible amyloids, termed yeast prions, are self-perpetuating heritable protein isoforms. Prion propagation in yeast is controlled by the chaperone machinery which includes Hsp104, Hsp70, and Hsp40 proteins. In this work, the yeast Saccharomyces cerevisiae is employed as a model to understand the molecular basis of how chaperones regulate the formation and propagation of amyloids. Here, I focused on the ribosome-associated chaperone Ssb, a member of the Hsp70 family, encoded by two genes (SSB1 and SSB2). My data indicate that during heat shock, Ssb is released from the ribosome and localized to the cytosol, interfering with Ssa, another Hsp70 protein, and impairing propagation of the prion [PSI+]. Attachment of Ssb1 to the activation domain of the protein Gal4, which contains a strong nuclear localization signal (AD-Ssb1), causes re-localization of Ssb to the nucleus. This leads to cytotoxicity and interference with propagation of [PSI+], [URE3], and [PIN+], prion forms of the Sup35, Ure2 and Rnq1 proteins, respectively. Moreover, relocation of the modified Ssb (AD-Ssb1) to the nucleus affects the function of Ssa directly or through a co-chaperone that is important for Ssa to function. I also found that deletion of ZUO1 (coding for the Hsp40 cochaperone of Ssb) increases spontaneous formation of the prion [URE3]. Moreover, in the absence of Ssb, mitotic stability of the prion forms of Ure2 ([URE3]) and Lsb2 ([LSB+]) is increased, while normally non-heritable mnemon aggregates of Ste18, [STE+] become heritable. De novo formation of [LSB+] and [STE+] is also increased in the absence of Ssb, especially during heat shock, which leads to massive accumulation of the [LSB+] prions. Overall, these results indicate that Ssb is a general anti-prion regulator, whose impact is not restricted only to the [PSI+] prion. In combination with the ribosome-associated chaperone complex (RAC), Ssb acts as a general modulator of cytosolic amyloid aggregation and can, directly or indirectly, repress prion generation and cure prions after they arise, counteracting prion toxicity. To further explore interactions among chaperones, prions and ribosomal machinery, I have constructed a yeast [PSI+] strain containing a deletion of all chromosomal rRNA genes (rdn1Δ), whose viability is dependent on a multicopy rRNA coding plasmid. This work expands the current knowledge of the role ribosome apparatus and ribosome-associated chaperones in heritable protein aggregation.