Feedback control of prion formation and propagation by the ribosome‐associated chaperone complex

Abstract

Summary Cross-beta fibrous protein aggregates (amyloids and amyloid-based prions) are found in mammals (including humans) and fungi (including yeast), and are associated with both diseases and heritable traits. The Hsp104/70/40 chaperone machinery controls propagation of yeast prions. The Hsp70 chaperones Ssa and Ssb show opposite effects on [PSI+], a prion form of the translation termination factor Sup35 (eRF3). Ssb is bound to translating ribosomes via ribosome-associated complex (RAC), composed of Hsp40-Zuo1 and Hsp70-Ssz1. Here we demonstrate that RAC disruption increases de novo prion formation in a manner similar to Ssb depletion, but interferes with prion propagation in a manner similar to Ssb overproduction. Release of Ssb into the cytosol in RAC-deficient cells antagonizes binding of Ssa to amyloids. Thus, propagation of an amyloid formed due to lack of ribosome-associated Ssb can be counteracted by cytosolic Ssb, generating a feedback regulatory circuit. Release of Ssb from ribosomes is also observed in wild type cells during growth in poor synthetic medium. Ssb is, in a significant part, responsible for the prion destabilization in these conditions, underlining the physiological relevance of the Ssb-based regulatory circuit.

To CURe or not to CURe? Differential effects of the chaperone sorting factor Cur1 on yeast prions are mediated by the chaperone Sis1

“…S7B). To find out whether or not Cur1 is able to bind prion aggregates directly, we performed a pulldown assay with His 6 -tagged Sup35NM as described previously (Kiktev et al, 2015). However, we could not detect any Cur1-YFP bound to Sup35/Sup35NM-His 6 aggregates (Supporting Information Fig.…”

“…150 ml of final culture was pelleted 4 h after copper addition. Cell lysis was performed as described (Kiktev et al, 2015). Cell lysates were pumped through Ni-NTA agarose resin for 1.5 h using the peristaltic pump (Bio-Rad) and eluted using the imidazole containing buffer.…”

“…Ydj1 also plays important role in propagation of [PIN 1 ] (Summers et al, 2009) and is essential for propagation of [SWI 1 ] (Hines et al, 2011). Deletion of another yeast Hsp40 protein, Zuo1, which is a component of by the ribosome-associated chaperone complex, antagonizes [PSI 1 ] propagation due to release of the other member of the Hsp70 family, Ssb, from the ribosome into cytosol, that leads to reduction in binding of Ssa to prion aggregates (Kiktev et al, 2015;Chernoff and Kiktev, 2016).…”

Combined tRNA modification defects impair protein homeostasis and synthesis of the yeast prion protein Rnq1

“…Interestingly, however, different defects of the ribosome associated chaperone complex (RAC) induced in zuo1, ssz1 and ssb1 ssb2 mutants all increased de novo generation of the [PSI C ] prion. [24][25][26] This may suggest that interference with translation at the level of tRNA functionality might impact on the tendency to form spontaneous amyloid aggregates as well, since similarities in protein homeostasis defects with the RAC mutants exist. However, dual tRNA modification loss affecting tRNA …”

Yeast prions are useful for studying protein chaperones and protein quality control

“…For example, specific J-proteins and Hsp70s associated with ribosomes cooperate to assist folding of nascent polypeptides, and alterations of these chaperones affect prion appearance or propagation. [56][57][58][59] Further work with this system could provide new insight into the functions of these chaperones. Prions are also affected by TPR-containing co-chaperones that bind the C-terminus of Hsp70 to regulate its activity and recruit it to other chaperone machines.…”