Three J-proteins impact Hsp104-mediated variant-specific prion elimination: a new critical role for a low-complexity domain

Abstract

Prions are self-propagating protein isoforms that are typically amyloid. In Saccharomyces cerevisiae, amyloid prion aggregates are fragmented by a trio involving three classes of chaperone proteins: Hsp40s, also known as J-proteins, Hsp70s, and Hsp104. Hsp104, the sole Hsp100-class disaggregase in yeast, along with the Hsp70 Ssa and the J-protein Sis1, is required for the propagation of all known amyloid yeast prions. However, when Hsp104 is ectopically overexpressed, only the prion [PSI⁺] is efficiently eliminated from cell populations via a highly debated mechanism that also requires Sis1. Recently, we reported roles for two additional J-proteins, Apj1 and Ydj1, in this process. Deletion of Apj1, a J-protein involved in the degradation of sumoylated proteins, partially blocks Hsp104-mediated [PSI⁺] elimination. Apj1 and Sis1 were found to have overlapping functions, as overexpression of one compensates for loss of function of the other. In addition, overexpression of Ydj1, the most abundant J-protein in the yeast cytosol, completely blocks Hsp104-mediated curing. Yeast prions exhibit structural polymorphisms known as "variants"; most intriguingly, these J-protein effects were only observed for strong variants, suggesting variant-specific mechanisms. Here, we review these results and present new data resolving the domains of Apj1 responsible, specifically implicating the involvement of Apj1's Q/S-rich low-complexity domain.

Keywords: Hsp104; Molecular chaperone; Prion; Protein misfolding; Yeast.

Fig. 1

Effect of Hsp104 expression on yeast prion propagation. Pink diamonds represent amyloid fibers, and red triangles represent soluble prionogenic protein. Deletion of the HSP104 gene results in the loss of all amyloid yeast prions, as does chemical inhibition of Hsp104 ATPase activity by guanidine hydrochloride. Overexpression of Hsp104 destabilizes [URE3] and results in the efficient curing of the prion [PSI⁺], but not other prions (Matveenko et al. 2018)

Fig. 2

J-protein primary structure diagrams. Summary of primary sequences of J-proteins and J-protein constructs used in this study. Protein regions are denoted as follows: J J-domain, G/F glycine/phenylalanine-rich region, G/M glycine/methionine-rich region, QS glycine-rich region also rich in glutamine and serine, ZBD zinc-binding domain, CTD I/II C-terminal peptide-binding domains I and II, D dimerization domain. Black line represents random peptide sequence with a C-terminal human influenza hemagglutinin (HA) tag

Fig. 3

Apj1 domain analysis for Hsp104-mediated elimination of strong [PSI⁺] variants. a Strong [PSI⁺]^STR and [PSI⁺]^Sc4 cells of the W303 genetic background lacking genomic APJ1 were transformed with plasmids expressing wild-type Apj1, Apj1-J-RHA, the first 161 residues of Apj1, or the Y/A chimera of Ydj1 and Apj1. Cells were then transformed with a plasmid overexpressing Hsp104 (GPD-HSP104) that normally cures [PSI⁺]. Color phenotype assays are shown for representative transformants (n = 10 for each variant). b Lysates of representative cells from panel A were resolved by SDD-AGE and subjected to immunoblot analysis using polyclonal antibody raised against Sup35. c Lysates of representative cells from panel A were resolved by SDS-PAGE and subjected to immunoblot analysis using polyclonal antibody raised against Apj1. Load control shown is a nonspecific protein cross-reacting with our Apj1 primary antibody