Investigation of the [ URE 31 Phenomenon in S . cerevisiae by

This project's first goal was to express and purify Ure2p in both its [ure3-0] and [URE3] conformations. To this end, a S. cerevisiae strain and galactose-inducible URE2 expression vectors were made, and by all tests they seemed suitable for this purpose. The prion model of disease states that PrPSc proteins, whether spontaneously formed or introduced from the outside, cause neurodegenerative disorders, including scrapie, bovine spongiform encephalopathy, and Creutzfeld-Jacob disease (reviewed in Brown et al, 1991; Prusiner, 1991). The prion model also maintains that the mammalian PrP protein can exist in two forms: a normal form (PrPc) and a prion form (PrPSc, see Figure 1). The PrPSc proteins are infectious because they convert the PrPc proteins into more PrPScs. The existence of prions has been controversial for decades. Most evidence supports the model, but a few critical experiments have not been successfully performed, presumably because working with the PrP system is difficult. In 1994, Wickner showed that prions might also exist in S. cerevisiae. The yeast genetic phenomena [PSI+ ] and [URE31 could be explained if [PSI+] and [URE3] were prion forms of the proteins Sup35 and Ure2 (Wickner, 1994). If yeast do have prions, then working with them might be easier than working with PrP prions. Through [PSI+] or [URE31, it might be possible to establish beyond reasonable doubt that prions exist. Mammalian prion research, to date, has several weak points. For instance, critics worry that the infectivity associated with purified PrPSc might be due to a contaminating nucleic acid. The only way to satisfy these critics is to make the PrP synthetically (or in an unrelated organism like E. coli), to purify it, convert it to its prion form, and then demonstrate its infectivity. For PrP, every step in this process has proved difficult. The PrP protein is too large to make synthetically. For unknown reasons, expressing it at reasonable levels has been hard (Mehlhorn et al., 1996), and has only recently been achieved (Riek et al., 1996). Converting the normal form of the protein INTRODUCTION to its prion form has also been challenging, and the best effort to date has produced so little putatively infectious protein that researchers can not detect a difference in infectivity after the conversion (Kocisco et al., 1994). In my work, I hoped to use S. cerevisiae, both to learn about yeast prions and to address some of the issues that remained unresolved with PrP. First of all, I wanted to purify the hypothetical yeast prion protein, Ure2p, in its prion and nonprion forms. In mammals, infectivity is assayed by injecting infectious material into an animal and waiting for it to get sick. I wanted to find a way of "injecting" possibly infectious Ure2 protein into yeast cells, and assay whether they were converted into prion strains. I also hoped to convert normal Ure2 protein to its prion form in vitro. Using either in vitro--converted Ure2p or synthetic Ure2-N terminus, I hoped to "infect" normal yeast cells, thus showing that pure protein is sufficient to convert the cells to their prion form. About URE2. Roughly twenty-five years ago, Francois Lacroute was investigating S. cerevisiae's uridine biosynthesis pathway (see Figure 2). Surprisingly, his ura2URA3 + cells failed to grown on ureidosuccinate (USA). Consequentially, he looked for mutations which would allow the cells to grow, and he discovered several. One type, he called ure2-. These cells had a single, recessive, nuclear mutation which permitted growth on USA. A second "mutation" was [URE31, a dominant, non-nuclear trait (Lacroute, 1971). Thus, [URE3] and ure2 cells have the same phenotype with regard to USA use, but the traits are genetically distinct. Later work by Coschigano and Magasanik (1991) resulted in the identification and cloning of URE2. By sequence, the Ure2 protein (Ure2p) is a 40 kD protein with Cterminal homology to glutathione-S-transferases. The protein's N-terminal 64 amino acids consist of 40% asparagine, and 20% serine and threonine. URE2 is involved in the regulation of S. cerevisiae's nitrogen metabolism; specifically, it is responsible for repression of Gln3p in response to a good nitrogen source, such as glutamine or ammonium (Coschigano and Magasanik, 1991, see Figure 3). Gln3p activates the transcription of a variety of genes, including DAL5, the allantoate permease (reviewed in Magasanik, 1992). This permease is USA's means of entry into the yeast cell (Magasanik, 1992). Therefore, non-functional URE2 permits growth on USA because its absence permits Gln3p to activate transcription of the USA permease. The prion connection. Wickner's groundbreaking paper (Wickner, 1994) proposed that the [PSI+] is a prion form of Sup35p, and that [URE31 is a prion form of Ure2p. To support this hypothesis, he used cytoduction to confirm the dominant, cytoplasmically-inherited nature of [URE31, and he demonstrated that the URE2 gene was necessary for [URE3] maintenance. He also showed that growing [URE31 cells on 5 mM guanidine hydrochloride effectively and reversibly cured cells of their [URE31 phenotype. In a second paper, Masison and Wickner (1995) showed that URE2's N-terminus is necessary for prion formation, while its C-terminus is necessary and sufficient for complementation. Consistent with the hypothesis that prion formation is a stochastic event whose probability depends on the amount of Ure2p, overexpression of URE2 increased the frequency at which cells "went prion" by 20-200 fold. Overexpression of the N-terminus increased the frequency to 3000 times the background level. Recent research on [PSI+] has, by analogy, yielded some exciting clues on a possible mechanism of [URE31 formation. Work by Chernoff et al., (1995), Paushkin et al. (1996), and Patino et al., (1996) strongly suggests that the prion form of Sup35p is an insoluble aggregate, with the N-terminal domain mediating aggregation. Certainly the prion form of PrP is an insoluble aggregate (Meyer et al., 1986, Gabizon et al., 1988), and aggregation could explain the increases in protease resistance for PrPSC and for Ure2p in [URE3] strains (Masison and Wickner, 1995). Come et al. (1993) suggested that prion formation might involve a nucleation-dependent, selfassembly mechanism. Consistent with this model is Glover and coworkers' (1997) finding that purified Sup35p precipitates into fibers with nucleation-propagationlike kinetics. MATERIALS and METHODS Vector construction. (a) Galactose-inducible expression vectors. I PCR-amplified URE2 with primers which gave it a 5' BamHI site, eliminated its stop codon, and gave it a 3' BspEI site followed by a SacI site. I inserted this fragment into the vector pBl-UASG (courtesy of Dr. Thomas Oehler, then in Dr. Leonard Guarente's lab, MIT), between its BamHI and SacI sites. This placed URE2 behind the galactose-inducible Gall10 promoter, producing pUASG-URE2. Just upstream of the UASG was a XhoI site from pBl-UASG's polylinker. I synthesized the oligo 5'GGTCCGGAGGTGGTGACTACAAGGACGACGATGA CAAGCACCATCATCACCATCACTGTTGAGTGACTGAGAGCTCGG-3' and its complement. This oligo encodes the FLAG epitope (Eastman Kodak Co., Rochester, NY), six histidines, and a C-terminal cysteine. I annealed and digested the oligos with BspEI and SacI. I ligated this fragment to the XmnI-BspEI portion of pUASGURE2 and the XmnI-SacI portion of pUASG-URE2. This triple ligation produced pUASG-URE2e. I inserted the XhoI-SacI fragment from pUASG-URE2e into pRS424 (Sikorski and Hieter, 1989), producing ET-URE2e. To make ET-URE2, I excised the tagged URE2 gene with BamHI and SacI, and replaced it with a PCR-amplified, untagged