Mick F Tuite — Page 2 | PubMed Health

PubMed2002

The Candida albicans Sup35p protein (CaSup35p): function, prion-like behaviour and an associated polyglutamine length polymorphism.

The Sup35p protein of Saccharomyces cerevisiae is an essential translation factor whose prion-like properties give rise to the non-Mendelian genetic element [PSI(+)]. In this study the SUP35 gene from the related yeast species Candida albicans has been characterized. The CaSUP35 gene encodes a protein (CaSup35p) of 729 aa which shows 65% amino acid identity to the S. cerevisiae Sup35p protein (ScSup35p), with the C-terminal region showing greater identity (79%) than the N-terminal region. The full-length CaSup35p can functionally replace ScSup35p in S. cerevisiae although complementation is only complete when CaSup35p is overexpressed. Complementation only requires expression of the CaSup35p C domain. In S. cerevisiae the full-length CaSup35p is unable to establish a prion-like aggregated state even in the presence of endogenous ScSup35p prion 'seeds', thus confirming the existence of a species barrier in fungal prion propagation. Subcellular localization studies in C. albicans show that although CaSup35p is normally ribosome-associated, when not ribosome-associated, it does not form pelletable high-molecular-mass aggregates characteristic of the ScSup35p in [PSI(+)] strains. Unlike the ScSup35p, the CaSup35p N domain contains a number of polyglutamine repeats although it does contain seven copies of the peptide GGYQQ that is repeated in the ScSup35p N domain. Analysis of the CaSUP35 gene from 14 different strains of C. albicans identified four naturally occurring polymorphisms associated with changes in the length of the largest of the polyglutamine repeats. These findings have important implications for the evolution of fungal prion genes.

Amino Acid Sequence↗

PubMed2002

Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast.

[PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.

Cell Division↗