Mutational analysis of Sse1 (Hsp110) suggests an integral role for this chaperone in yeast prion propagation in vivo

Abstract

The yeast Hsp110 chaperone Sse1 is a conserved protein that is a noncanonical member of the Hsp70 protein superfamily. Sse1 influences the cellular response to heat stress and has also been implicated in playing a role in the propagation of prions in yeast. Sse1 can seemingly exert its effects in vivo through direct or indirect actions by influencing the nucleotide exchange activity of canonical cytosolic Hsp70s. Using a genetic screen based on the inability to propagate the yeast [PSI(+)] prion, we have identified 13 new Sse1 mutants that are predicted to alter chaperone function through a variety of different mechanisms. Not only are these new Sse1 mutants altered in the ability to propagate and cure yeast prions but also to varying degrees in the ability to grow at elevated temperatures. The expression levels of chaperone proteins known to influence yeast prion propagation are unaltered in the Sse1 mutants, suggesting that the observed phenotypic effects are caused by direct functional alterations in these mutants. Mapping the location of the mutants onto the Sse1 crystal structure suggests that more than one functional alteration in Sse1 may result in changes in prion propagation and ability to function at elevated temperatures. All Sse1 mutants isolated provide essential functions in the cell under normal growth conditions, further demonstrating that essential chaperone functions in vivo can to some degree at least be detached from those related to propagation of prions. Our results suggest that Sse1 can influence prion propagation through a variety of different mechanisms.

Keywords: Hsp110; Hsp70; Saccharomyces cerevisiae; Sse1; chaperone; nucleotide exchange factor; prion.

Figure 1

(A) Sse1 mutants that impair prion propagation are located in various domains of the protein. Numbers above refer to amino acids that define the boundaries of the nucleotide-binding domain (NDB), linker region (L), substrate-binding domain (SBD), Hsp110 insertion region (I), and Hsp110 extension region (E). Mutants isolated that impair prion propagation are indicated below the linear structure. (B) Phenotype of Sse1 mutants that impair prion propagation. Top panel shows color on YPD, middle panel depicts growth on medium lacking adenine, and bottom panel is growth on YPD at 39°.

Figure 2

Sse1 mutants exhibit a complex growth phenotype when grown on medium lacking adenine. The absence of histidine and the presence of FES1 can affect the ability of Sse1 mutants to grow on medium lacking adenine. Top section is growth in presence of either vector control or overexpression of CIA1, and bottom section is in the presence of over-expressed FES1. The results shown are representative of three independent experiments, for controls this constitutes two experiments with vector only and one with CIA1 overexpression.

Figure 3

No change in protein levels of chaperones known to alter [PSI⁺] propagation in Sse1 mutants. Western blot analysis to measure protein levels of Sse1, Hsp70 (Ssa), and Hsp104. After initial blotting with anti-Sse1 antisera, the membrane was stripped and subsequently probed with Hsp104 and Hsp70 antibodies. The membrane was stained with Amido Black to show loading.

Figure 4

Mapping of mutations onto Sse1 structure. (A) Structural model of Sse1 (PDB: 2QXL) with the residues of interest highlighted and in ball and stick format. Domains are colored to correspond to Figure 1A. Images were generated using Pymol (DeLano 2002).

Figure 5

Phenotypic analysis of yeast cells expressing Sse2 as the sole source of Hsp110. Growth of Sse1, Sse2, and Sse2 derived mutants on medium lacking adenine (top growth panels) and at elevated temperature (lower growth panels). Western blotting was used to assess expression levels of Sse1, Sse2, and mutants (bottom panels).

Figure 6

Complementation of sse1 sse2 deletion strain by overexpression of FES1 or mammalian HSPH1. Growth of sse1 sse2 expressing FES1 or HSPH1 in place of SSE1 was assessed in two strain backgrounds; CMY02 (G600 background, left section) and CMY03 (BY background, right section). As expected, vector only control produced no growth in either background.