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Destruction Or Potentiation of Different Prions Catalyzed by Similar Hsp104 Remodeling Activities

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Destruction Or Potentiation of Different Prions Catalyzed by Similar Hsp104 Remodeling Activities Molecular Cell 23, 425–438, August 4, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.molcel.2006.05.042 Destruction or Potentiation of Different Prions Catalyzed by Similar Hsp104 Remodeling Activities James Shorter1 and Susan Lindquist1,* transitions of prions and amyloids are profoundly 1 Whitehead Institute for Biomedical Research modulated by molecular chaperones and protein- 9 Cambridge Center remodeling factors (Tuite and Cox, 2003; Muchowski Cambridge, Massachusetts 02142 and Wacker, 2005). However, despite intense scrutiny from many cutting-edge laboratories worldwide, there is still not yet a single example in which the mechanistic Summary interplay between chaperones/protein-remodeling fac- tors and amyloid/prion biochemistry and biology is Yeast prions are protein-based genetic elements that well understood. Here, we address these questions for self-perpetuate changes in protein conformation and two prions of Saccharomyces cerevisiae: [PSI+], whose function. A protein-remodeling factor, Hsp104, con- protein determinant is Sup35, and [URE3], whose pro- trols the inheritance of several yeast prions, including tein determinant is Ure2. Following standard nomencla- those formed by Sup35 and Ure2. Perplexingly, dele- ture, italicized capital letters denote dominant genetic tion of Hsp104 eliminates Sup35 and Ure2 prions, elements, and brackets indicate a non-Mendelian mode whereas overexpression of Hsp104 purges cells of of inheritance. Both properties stem from the ability of Sup35 prions, but not Ure2 prions. Here, we used prions to store and transmit biological information via pure components to dissect how Hsp104 regulates alternative, self-perpetuating structures and functions. prion formation, growth, and division. For both The genetic trait conferred by [PSI+] is due to the con- Sup35 and Ure2, Hsp104 catalyzes de novo prion nu- version of a translation termination factor, Sup35, to cleation from soluble, native protein. Using a distinct a nonfunctional prion state (Tuite and Cox, 2003). mechanism, Hsp104 fragments both prions to gener- [PSI+] causes ribosomes to readthrough stop codons. ate new prion assembly surfaces. For Sup35, the frag- This unleashes hidden genetic variation that confers mentation endpoint is an ensemble of noninfectious, phenotypic diversity and selective advantages in di- amyloid-like aggregates and soluble protein that can- verse settings (True and Lindquist, 2000; True et al., not replicate conformation. In vivid distinction, the 2004). The ability of Sup35 to form [PSI+] has been con- endpoint of Ure2 fragmentation is short prion fibers served for hundreds of millions of years (Nakayashiki with enhanced infectivity and self-replicating ability. et al., 2001) and is almost certainly due to the advanta- These advances explain the distinct effects of geous phenotypic plasticity and evolvability it imparts Hsp104 on the inheritance of the two prions. (Shorter and Lindquist, 2005; but see Nakayashiki et al. [2005]). Introduction Sup35 has three functionally distinct domains. The C-terminal GTPase domain (C) (amino acids 254–685) The breadth of biological phenomena attributable to confers translation termination activity (Tuite and Cox, prion proteins has greatly expanded and now encom- 2003). The N-terminal, glutamine/asparagine-rich do- passes self-replicating agents of infectivity, inheritance, main (N, amino acids 1–123) drives the switch from the and potentially long-term memory formation (Shorter soluble, functional [psi2] state to the insoluble [PSI+] and Lindquist, 2005). Prions switch between profoundly prion state (Paushkin et al., 1996; Patino et al., 1996). different structural and functional conformations. Criti- The highly charged middle domain (M, amino acids cally, at least one of these conformations self-replicates 124–253) promotes solubility in the [psi2] state and en- by templating the conversion of other conformers of the sures [PSI+] stability through cell division (Liu et al., same protein to the self-replicating form. Prions transmit 2002). spongiform encephalopathies in mammals (Shorter and [URE3] alters the way yeast utilize nitrogen sources Lindquist, 2005). However, prions also function as pro- (Wickner et al., 2004). Its protein determinant, Ure2, neg- tein-based genetic switches. The propagation of func- atively regulates a suite of proteins that import and tionally distinct conformers produces distinct, often ad- catabolize secondary nitrogen sources. When Ure2 con- vantageous, heritable phenotypes (True and Lindquist, verts to its dysfunctional prion form, yeast cells use poor 2000; True et al., 2004). Such self-replicating states nitrogen sources even when rich ones are available may function to store and transmit information, includ- (Wickner et al., 2004). Ure2 has a C-terminal functional ing long-term memory in neurons (Si et al., 2003; Shorter domain (amino acids 81–354) and an N-terminal aspara- and Lindquist, 2005). gine-rich domain (amino acids 1–80) that is necessary Different prions have unrelated sequences and func- and sufficient for prion formation and propagation tions, but all form self-propagating, amyloid fibers under (Wickner et al., 2004). [URE3] is not as well conserved physiological conditions (Shorter and Lindquist, 2005). as [PSI+] but may sometimes be advantageous (Talarek These fibers are akin to those connected with several et al., 2005; Shorter and Lindquist, 2005; but see Na- devastating neurodegenerative disorders (Muchowski kayashiki et al. [2005]). and Wacker, 2005). However, other amyloids function Sup35 and Ure2 form prions once their N-terminal do- as specialized cellular nanostructures (Wickner et al., mains switch from soluble, largely unstructured states 2004; Shorter and Lindquist, 2005). The conformational to b sheet-rich amyloid forms that rapidly convert solu- ble protein to the same state (Glover et al., 1997; Taylor *Correspondence: [email protected] et al., 1999). In vitro, the N and M domains of Sup35 (NM) Molecular Cell 426 form amyloid fibers after a lag phase. Fibers elongate and a panel of Hsp104 mutants, we find extraordinary from both ends, catalyzing the conformational conver- congruencies in the ways that Hsp104 remodels the sion of soluble protein (Serio et al., 2000; Krishnan and conformations of these two unrelated proteins, includ- Lindquist, 2005). [psi2] cells can be transformed to the ing the mechanisms by which (1) Hsp104 promotes [PSI+] state by NM fibers (Tanaka et al., 2004). Thus, fi- prion assembly, (2) high Hsp104 concentrations restrict bers harbor prion infectivity. Similarly, Ure2 fibers as- access to the prion state, and (3) Hsp104 deconstructs semble after a lag phase, seed the assembly of soluble prions. Despite these remarkable and unexpected simi- Ure2, and transform [ure-o] cells to the [URE3] state larities in how Hsp104 regulates these different prions, (Taylor et al., 1999; Fay et al., 2003; Zhu et al., 2003; we also elucidate a critical singularity that explains Brachmann et al., 2005). why Hsp104 overexpression cures [PSI+] but not [PSI+] and [URE3] are faithfully inherited, with a fre- [URE3]. quency of loss of w1025–1027 (Tuite and Cox, 2003). Their formation and inheritance depend absolutely on Results Hsp104, a protein-remodeling factor (Chernoff et al., 1995; Moriyama et al., 2000). Hsp104 rescues cells Spontaneous Assembly of Sup35 and Ure2 Fibers from environmental stress by wresting denatured, ag- First, we examined whether full-length Sup35 can con- gregated proteins apart and reactivating them (Parsell vert from the soluble to the fibrous state. The C-terminal et al., 1994b; Glover and Lindquist, 1998). Hsp104 is an domain of Sup35, which contains four GTP binding AAA+ (ATPases associated with diverse activities) pro- modules, made the protein prone to amorphous non- tein (Hanson and Whiteheart, 2005) that hexamerizes prion aggregation (data not shown). To reduce this prob- in the presence of ADP or ATP (Parsell et al., 1994a). lem, the protein was purified under native conditions in Each protomer has two AAA+ modules (nucleotide bind- the constant presence of GTP, and GTP was retained ing domains, NBD1 and NBD2) separated by a coiled- in all assembly reactions. Sup35 was analyzed immedi- coil middle domain and flanked by N- and C-terminal do- ately after purification, as freezing and thawing caused mains. During substrate remodeling, the middle domain denaturation. Further, we eliminated proteolytic frag- of Hsp104 undergoes cycles of conformational change ments harboring the prion domain, which influenced as- driven by the cooperative ATPase activities of both sembly kinetics (data not shown). Fibrillization of native NBDs (Hattendorf and Lindquist, 2002; Cashikar et al., Sup35 was analyzed by SDS solubility, Thioflavin-T 2002). (ThT) fluorescence, and electron microscopy (EM). All Deletion of Hsp104 eliminates [PSI+] and [URE3] three assays gave very similar results throughout. (Chernoff et al., 1995; Moriyama et al., 2000), as do spe- Newly prepared Sup35 formed fibers spontaneously cific point mutations in the NBDs of Hsp104, and growth after an w2 hr lag phase, TL (time elapsing before the in the presence of 5 mM guanidium chloride (GdmCl), an first detection of amyloid; Figures 1A and 1B, and see uncompetitive inhibitor of Hsp104 ATPase activity (Pat- Table S1 in the Supplemental Data available with this ar- ino et al., 1996; Wegrzyn et al., 2001; Hattendorf and ticle online). TL was much longer for Sup35 than for the Lindquist, 2002; Ripaud et al., 2003; Grimminger et al.,
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