Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Abstract

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI+] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN+] prions was less sensitive than [URE3], but more sensitive than [PSI+]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.

Reconstitution of a Mycobacterium tuberculosis proteostasis network highlights essential cofactor interactions with chaperone DnaK

“…In recruiting aggregates to DnaK and its homologs, J proteins are proposed to act upstream of ClpB homologs (Hsp104) (Fig. 1B) (51,52). Higher eukaryotes lack cytosolic ClpB homologs (37), and it was shown that when purified metazoan J proteins from two different protein classes were mixed, they could disaggregate denatured proteins in vitro (38).…”

The Role of the Hsp40 Chaperone Sis1 in Yeast Prion Propagation

“…[30]. These findings were confirmed and expanded upon in 2014 [33]. Reidy and coauthors first expanded upon their earlier findings that Sis1 was the Hsp40 working with the E. coli Hsp100, Hsp70 and nucleotide exchange factor (ClpB, DnaK and GrpE, respectively) in prion propagation and Ydj1 directed the same disaggregation machinery to heat stress-induced aggregates.…”

“…The Higurashi model of Ydj1-mediated curing of [URE3] was strengthened by findings in Sis1-Ydj1 chimera paper that further characterized [URE3]'s strong dependence on Sis1 [33].…”

“…They then employed the suite of Sis1 domain truncations used in the Yan, Kirkland and Harris papers to determine the specific requirements for Sis1 on [URE3] propagation, information that was lacking. Remarkably, [URE3] was lost or greatly destabilized by any of the mutations in Sis1 that were tested [33]. In some cases, the prion could be selected for and maintained in the presence of a particular Sis1 truncation, such as deletion of the dimerization motif, by growth on media-lacking adenine.…”

“…Whether the essential function of Sis1 is also required for prion propagation is unclear, as will be discussed. The nonessential functions of Sis1 can easily be studied using yeast prions, because the different yeast prions all seem to have differing requirements of Sis1 [30][31][32][33]. It is these nonessential Sis1 functions that are the focus of this chapter.…”