Mammalian Prion Protein Suppresses Bax-induced Cell Death in Yeast

Aimin Li,David A Harris

doi:10.1074/jbc.c500058200

Abstract

Several lines of evidence suggest that PrP(C), the non-infectious form of the prion protein, may function to protect neurons and other cells from stress or toxicity. In this paper, we report on the use of the yeast Saccharomyces cerevisiae as a model system to assay the cytoprotective activity of PrP(C). The mammalian pro-apoptotic protein, Bax, confers a lethal phenotype when expressed in yeast. Since overexpression of PrP(C) has been found to prevent Bax-mediated cell death in cultured human neurons, we explored whether PrP could also suppress Bax-induced cell death in yeast. We utilized a form of mouse PrP containing a modified signal peptide that we had previously shown is efficiently targeted to the secretory pathway in yeast. We found that this PrP potently suppressed the death of yeast cells expressing mammalian Bax under control of a galactose-inducible promoter. In contrast, cytosolic PrP-(23-231) failed to rescue growth of Bax-expressing yeast, indicating that protective activity requires targeting of PrP to the secretory pathway. Deletion of the octapeptide repeat region did not affect the rescuing activity of PrP, but deletion of a charged region encompassing residues 23-31 partially eliminated activity. We also tested several PrP mutants associated with human familial prion diseases and found that only a mutant containing nine extra octapeptide repeats failed to suppress Bax-induced cell death. These findings establish a simple and genetically tractable system for assaying a putative biological activity of PrP(C).

Highlights

Of nucleic acid [2, 3]
We have demonstrated that PrP targeted to the secretory pathway of S. cerevisiae is capable of rescuing the lethal phenotype caused by heterologous expression of Bax, a pro-apoptotic member of the Bcl-2 gene family
Bax is a cytoplasmic protein that is translocated to mitochondria in response to apoptotic signals, where it promotes cell death by mediating release of cytochrome c, which in turn activates caspase-dependent pathways [29]

Summary

The abbreviations used are

PrP, prion protein; PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; BI-1, Bax inhibitor 1; DPAPB, dipeptidyl aminopeptidase B; GPD, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; SD, synthetic dextrose (glucose) medium; SG, synthetic galactose medium; WT, wild-type. PrP overexpression rescues cultured neurons and some mammalian cell lines from pro-apoptotic stimuli, including Bax expression, serum withdrawal, and cytokine treatment (9 –12). We tested whether PrP could rescue yeast from cell death induced by the pro-apoptotic protein, Bax, similar to the way PrP has been shown to protect cultured human neurons from Bax-induced death [9, 12]. We capitalized on our recent demonstration that PrP with a modified signal peptide is efficiently targeted to the yeast secretory pathway, where it becomes glycosylated, glycolipidanchored, and localized to the plasma membrane as it does in mammalian cells [21]. We show here that PrP targeted to the secretory pathway efficiently suppresses Bax-induced cell death in yeast, and we perform a structure-function analysis to determine which features of the PrP molecule are essential for this effect.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION