Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. 2011 Feb 15; 108(7): 2999–3004.
Published online 2011 Jan 26. doi:  10.1073/pnas.1019368108
PMCID: PMC3041084

Protein-only mechanism induces self-perpetuating changes in the activity of neuronal Aplysia cytoplasmic polyadenylation element binding protein (CPEB)


Neuronal cytoplasmic polyadenylation element binding protein (CPEB) plays a critical role in maintaining the functional and morphological long-lasting synaptic changes that underlie learning and memory. It can undergo a prion switch, but it remains unclear if this self-templating change in protein conformation is alone sufficient to create a stable change in CPEB activity: a robust “protein-only” biochemical memory. To investigate, we take advantage of yeast cells wherein the neuronal CPEB of Aplysia is expressed in the absence of any neuronal factors and can stably adopt either an active or an inactive state. Reminiscent of well-characterized yeast prions, we find that CPEB can adopt several distinct activity states or “strains.” These states are acquired at a much higher spontaneous rate than is typical of yeast prions, but they are extremely stable—perpetuating for years—and have all of the non-Mendelian genetic characteristics of bona fide yeast prions. CPEB levels are too low to allow direct physical characterization, but CPEB strains convert a fusion protein, which shares only the prion-like domain of CPEB, into amyloid in a strain-specific manner. Lysates of CPEB strains seed the purified prion domain to adopt the amyloid conformation with strain-specific efficiencies. Amyloid conformers generated by spontaneous assembly of the purified prion domain (and a more biochemically tractable derivative) transformed cells with inactive CPEB into the full range of distinct CPEB strains. Thus, CPEB employs a prion mechanism to create stable, finely tuned self-perpetuating biochemical memories. These biochemical memories might be used in the local homeostatic maintenance of long-term learning-related changes in synaptic morphology and function.

Keywords: functional amyloid, molecular memory, synapse maintenance, translational activation

The mammalian prion protein, PrP, can adopt an alternative self-perpetuating conformation that causes transmissible neurodegenerative diseases (1). In fungi, unrelated proteins adopt similar self-perpetuating prion conformations that serve as “protein-only” elements of epigenetic inheritance (2). Most fungal prion proteins can exist in two structurally and functionally distinct conformations, one soluble and the other a self-templating fibrillar amyloid (25). These self-templating amyloid states are passed from mother cells to their daughters via the cytoplasm, resulting in the inheritance of prion phenotypes that do not follow the Mendelian laws of inheritance. Notably, many yeast prions populate an ensemble of related but distinct amyloid states, each of which faithfully self-templates, which thereby produce a series of related phenotypes with distinct intensities.

Prion amyloids are not normally toxic in yeast. Indeed, depending upon the growth conditions, the phenotypes conferred by yeast prions can produce strong growth advantages (5, 6). In the human brain, amyloids are generally associated with fatal neurodegenerative diseases, but it is now widely believed that in these conditions, the amyloid is itself a benign, even protective, state with toxicity caused by other types of misfolded conformers (7). Indeed, amyloids are now known to play beneficial roles in several normal biological processes in diverse organisms, including cell-adhesion, skin pigmentation, and peptide hormone storage (4, 5, 812). A particularly exciting possibility for an amyloid function is one suggested for the neuronal isoform of Aplysia CPEB (cytoplasmic polyadenylation element binding protein): that this protein uses a prion-like amyloid switch to create a molecular memory at neuronal synapses, thereby establishing a long-lasting mark for synapse maintenance (1317).

CPEBs are found in many cell types and regulate the translational dormancy and activation of specific mRNAs (18, 19). They bind U-rich cytosolic polyadenylation elements (CPEs) in mRNA 3′ UTRs and subsequently recruit the polyadenylation machinery. The neuronal version of CPEB localizes in the presynaptic terminal of Aplysia and the dendrites of mice, where it can be activated following synaptic stimulation (15). Active CPEB then elongates polyadenylated tails of CPE-containing mRNAs, which encode structural and regulatory proteins that maintain long-term synaptic growth. The neuronal isoforms of CPEB differ from that found in other cell types in having a glutamine-rich N-terminal extension similar to the prion-determining domains of yeast prions (17).

In yeast, Aplysia CPEB can exist either in an active or an inactive form (17). When CPEB is active, it induces the translation of a reporter mRNA with a CPE element in its 3′ UTR. This form of CPEB is in a larger protein/RNA complex than the inactive form and is dominant in crosses, suggesting that it might be a prion form of CPEB. This hypothesis came as a surprise because yeast prions are generally inactive in their amyloid conformations. The notion that the translationally active form of CPEB is prion-like gained support from a recent study in which overexpressed Aplysia CPEB formed self-templating multimers of an amyloid-nature in the Aplysia neuron (15). Aplysia CPEB is active at the synapse, where it binds to CPE-containing mRNAs (18). It remains to be determined whether CPEB multimers actually represent translationally active sites in the neuron. However, conversion to the multimeric state is enhanced by the relevant neurotransmitter, and blocking this conversion (with a multimer-specific antibody) interferes with the maintenance of long-term synaptic facilitation. Furthermore, the Drosophila CPEB homolog orb2 is required for long-term conditioning of male courtship behavior. Like Aplysia CPEB, Orb2 carries an N-terminal glutamine-rich sequence. Deletion of this domain impairs long-term memory formation (20), indicating a physiological role for this prion domain-like sequence.

Despite these supporting data, the notion that a switch to a self-templating amyloid polymer could serve as biochemical memory in the synapse was so unexpected that it continues to be viewed with considerable skepticism. The importance of understanding the mechanisms of synaptic memory demands a high standard of proof. Is a conformational switch in the CPEB protein alone sufficient to create a stable self-perpetuating change in CPEB activity and thereby form a protein-only molecular memory? This question is analogous to the long-standing controversy about whether self-perpetuating conformational changes in mammalian PrP were alone sufficient to create the transmissible agent in the spongiform encephalopathies. Confirming the protein-only hypothesis required generating prion conformers in vitro from purified protein and using this protein to transmit disease (21). Analogous experiments in which recombinant prion conformers transformed yeast cells, transmitting heritable new phenotypes, confirmed the prion mechanism for several endogenous yeast proteins (2, 2224).

Yeast cells are 1 billion years removed from Aplysia evolutionarily and do not have the synaptic environment normally involved in regulating CPEB's neuronal activities. Therefore, yeast cells provide a “living test tube” to investigate the intrinsic capacity of heterologously expressed Aplysia CPEB to act as a protein-only molecular memory. Using yeast, we demonstrate that CPEB has an ability to exist in several related but distinct self-perpetuating activity states (strains) that are, indeed, based on a protein-only prion mechanism.


CPEB Can Adopt Distinct Heritable Activity States.

In yeast, the activity of neuronal Aplysia CPEB can be readily assayed with a β-gal reporter mRNA carrying a CPE sequence in its 3′ UTR (17, 25). When CPEB is active yeast cells turn blue in the presence of the substrate X-Gal. When CPEB is inactive, β-gal is not expressed and cells remain white (Fig. 1A). Previous studies in yeast used a 2 μ vector with a highly variable copy number in cells that also produced a red pigment. To obtain much more uniform levels of CPEB expression and to more clearly visualize its activity state, we used a single copy CEN vector in cells without the red pigment. In this case, transformants were either white or blue on X-Gal plates. However, blue colonies were of many different shades (Fig. 1B). Such differences in color were not previously remarked upon.

Fig. 1.
The activity of Aplysia CPEB in yeast. (A) Neuronal Aplysia CPEB acts as a translational activator in its putative prion conformation. Yeast cells carrying soluble inactive CPEB and a β-gal reporter followed by a CPE are translationally dormant ...

After transformation, the ratio of cells in the CPEB-active versus the CPEB-inactive state was strongly influenced by the growth phase. Transformation of early log-phase cultures produced roughly equal mixtures of blue and white colonies; late log-phase cultures produced mostly blue colonies. This result might reflect some early influence of metabolism, as yeast cells switch from fermentation to respiration during this growth transition, or the effects of chaperone proteins, which are induced in late log. In any case, for both early and late log-phase transformants, blue colonies always exhibited many different shades of color.

To ensure that these differences in colony color reflect differences in CPEB activity, and not some artifactual difference in growth on X-Gal plates or substrate permeability, we assessed β-gal activity with the water-soluble substrate CPRG (chlorophenol red-β-d-galactopyranoside) in detergent-permeabilized cells (Fig. 1B, Lower). Here, the intensity of red color reflects the activity of the enzyme. Many different levels of CPEB activity were observed with suspensions from different individual colonies, and these correlated with the intensities of blue color in the original colonies on X-Gal.

Distinct CPEB Activity States Are Stably Inherited and Have a Prion-Like Genetic Character.

The stable inheritance of distinct but related phenotypic states, known as strains, over thousands of generations, is a common feature of prions in yeast. We continuously streaked cells with different CPEB activities biweekly over the course of a year or more. Switches in colony color occurred in both directions, but they were rare (Fig. S1). Activity states were stable for thousands of generations, with each cell giving rise to millions of progeny, and transitioning continuously through very different metabolic states: from rapid growth with abundant nutrients to stationary phase starvation. Thus, although a variety of CPEB activity states are accessible during the initial transformation, once a particular state is established: it is extremely stable and virtually impervious to changes in metabolism.

Another characteristic of prions in yeast is the dominance of their phenotypes in genetic crosses with nonprion strains. This dominance is because the protein template brought in by the prion-containing partner is equally efficient at templating the protein of both parents. To test the dominance of CPEB states, we mated cells from white colonies to cells from colonies with various shades of blue (hereafter “white cells” and “blue cells”). Matings between two white cells always produced white cells. Matings between white and blue cells always produced blue cells, with a color intensity true to that of the blue parent (Fig. S2).

As nonchromosomal, protein-based genetic elements, prions can be “donated” to mating partners by cytoduction (25), a process in which cytoplasmic factors (such as organelles or prions) are transferred from one cell to another without nuclear exchange. White, light blue, blue, and dark blue donors (the same strains as in Fig. 1B) were mated to white recipients. These recipients had both a defect nuclear fusion defect (KarΔ1–15) (Fig. 1C) and a mitochondrial defect (ρ0) that prevented growth on glycerol. Haploid progeny of nuclear fusion-deficient diploids were selected for the nuclear marker of the recipient (Ura+) and the cytoplasmic marker of the donor (by growth on glycerol without uracil). All lacked the His+ marker of the donor, confirming the lack of nuclear transfer. When replicated to X-Gal plates, they exhibited various shades of blue, all faithfully reflecting the distinct CPEB activity state of the donor (Fig. 1C, Right). Control cytoductions with white donors produced only white progeny, confirming that mating itself did not change CPEB activity. Thus, as for yeast prion strains, the distinct self-perpetuating activity states of CPEB are not because of nuclear polymorphisms but to stable cytoplasmically inherited traits (5). Following yeast nomenclature, we designate these states [CPEB+], with the brackets indicating non-Mendelian inheritance, and the capital letters and italics designating a dominant heritable element.

[CPEB+] Strains Can Template Distinct Self-Perpetuating Amyloid States.

A further characteristic of prion proteins is the modular nature of their prion domains. This nature allows them to template their distinct conformational states to other proteins that share the same prion domain, but no other amino acid sequence homology (4). The putative prion domain of CPEB, CPEB-Q, is an N-terminal, 128 amino acids long, and has a glutamine-rich composition reminiscent of yeast prion domains (17). Indeed, a glucocorticoid receptor transcription factor (GR) fused to the CPEB-Q domain spontaneously adopts active or inactive states that self-perpetuate as expected for a modular prion domain (17).

Using the single-copy constitutive expression constructs that allowed us to maintain and monitor distinct [CPEB+] strains so effectively, we were unable to physically detect the protein, either by Western blotting or immunofluorescence. Instead, to determine whether distinct [CPEB+] states are associated with distinct physical properties, we asked if they could template those states to a CPEB-Q-EGFP fusion protein. The CPEB-Q domain is very amyloidogenic and occasionally caused off-pathway nonamyloid aggregation in such fusions. Therefore, we included the highly charged M domain of the yeast prion protein Sup35, which counterbalances the aggregation tendency of its own prion domain but is otherwise not involved in prion formation (26). Cells with different CPEB activity states were transformed with a galactose-inducible CBEB-Q-M-EGFP on a 2 μ plasmid.

When induced with galactose, the fusion protein never produced foci in cells expressing CPEB-Q-M-EGFP alone (Fig. 2A) nor in white cells expressing full-length CPEB protein (Fig. 2B). The protein did produce foci in blue cells (Fig. 2 C and D). The variable copy number of the 2 μ CPEB-Q-M-EGFP vector precluded quantitative assessment and some cells were dark because they had lost the plasmid. However, consistently, large foci were observed in dark blue cells; smaller and fewer foci were observed in light blue cells, with some faint diffuse fluorescence both in cells with and without foci. A galactose-induced control without the putative prion domain, M-EGFP, never formed foci (27).

Fig. 2.
CPEB strains template distinct self-perpetuating amyloid states. (A) CPEB-Q-M-EGFP is under a galactose promoter on a 2 μ plasmid. It remains soluble when overexpressed for 6 h in w303 ADE+ cells lacking full-length CPEB. (B) Overexpressed CPEB-Q-M-EGFP ...

Most known prion proteins adopt an SDS-resistant amyloid conformation in the prion state. To determine whether these in vivo-templated foci were SDS-resistant amyloids, whole-cell lysates were separated using semidenaturing detergent agarose gel electrophoresis (SDD-AGE) (3, 28) and probed with an anti-GFP antibody (Fig. 2E). The CPEB-Q-M-EGFP protein from white cells ran at the position of the soluble nonamyloid monomer; the protein from blue cells ran as an SDS-resistant amyloid polymer. The polymers of light blue and dark blue cells were of similar size, but a greater fraction of the protein from dark blue cells migrated at this position (Fig. 2E). Thus, preexisting CPEB conformers in blue strains template an amyloid state to the CPEB-Q domain of newly synthesized protein, with strain-specific efficiencies.

Whole-Cell Lysates Seed Amyloid Formation by Purified CPEB-Q-M in a Strain-Specific Manner.

The efficiencies with which cell lysates with well-characterized yeast prions seed the polymerization of soluble recombinant prion protein vary in a strain-specific manner (29). We asked whether whole-cell lysates (0.7% vol/vol) from our white, light blue, and blue strains can seed the aggregation of recombinant CPEB with strain-specific characteristics. The protein was purified under denaturing conditions (6 M guanidine hydrochloride, GdnHCl) to remove any preexisting structure. We used CPEB-Q-M for these studies because full-length CPEB produces amorphous aggregates when so denatured.

Purified CPEB-Q-M diluted into assembly buffer remained soluble in quiescent reactions over the course of 18 h (Fig. 3A, first panel). Lysates of cells not expressing CPEB had no effect (Fig. 3A, second panel). Lysates of strains expressing CPEB caused a fraction of purified CPEB-Q-M to assemble into SDS-resistant amyloid (Fig. 3A, third to fifth panels). This amyloid-templating activity was strongest with lysates from dark blue cells. Assembly was, however, always incomplete; higher concentrations of lysate did not result in more assembly. We note that seeding the polymerization of bona fide yeast prion proteins with whole-cell lysates is also generally incomplete (29, 30). The efficiency we obtained with [CPEB+] was lower than characteristically obtained with the Sup35 prion, but not much lower than obtained with another well-characterized yeast prion, Rnq1 (30).

Fig. 3.
Whole-cell lysates seed recombinant CPEB-Q-M amyloid assembly in a strain-specific manner. (A) Lysates of light blue and blue strains constitutively expressing CPEB and overexpressing CPEB-Q-M-EGFP seed amyloid assembly of purified his6-tagged CPEB-Q-M. ...

We asked whether the aggregates generated in such primary seeding reactions could be propagated to purified CPEB-Q-M in a secondary seeding reaction. This result would provide further evidence that the seeding templates have a protein-only nature. The very small amount of aggregation observed with white cells could not be propagated. The aggregation activities from light blue and dark blue cells could be propagated (Fig. 3B), but again, reactions did not go to completion. Presumably, the remaining protein spontaneously adopts an off-pathway conformation that does not productively interact with the template. Again, similar results have been reported for Rnq1 (30). In vivo, chaperones and other factors likely act to maintain these proteins in conformations that permit more efficient templating.

CPEB-Q-M Fibers Are Sufficient to Induce Self-Perpetuating Changes in CPEB Activity.

The “gold standard” for establishing that a protein is a prion is to assemble recombinant, purified protein in vitro into a protein-only template that can transform cells from the nonprion to the prion state (31). To generate CPEB prion conformers in vitro solely from recombinant protein, we used purified guanidine-denatured CPEB-Q and CPEB-Q-M. Purified yeast prion domains spontaneously form prion fibers most efficiently under gentle agitation. When purified CPEB-Q and CPEB-Q-M were diluted into assembly buffer and subjected to gentle agitation, both formed insoluble material that was resistant to solubilization by SDS, as expected for an amyloid (Fig. 4A and Fig. S3). This material produced the same spectral shift on staining with Thioflavin T as observed with other amyloids. By transmission electron microscopy (TEM) (Fig. 4 B and C), CPEB-Q-M formed fibers that were more homogeneous than CPEB-Q, suggesting that the addition of the solubilizing M domain of Sup35 decreases off-pathway, nonamyloid aggregation and supports more efficient templating. In full-length CPEB, the RNA binding domain likely serves a similar function (perhaps with associated RNA). To determine whether the CPEB-Q and CPEB-Q-M fibers are fully functional prion replication templates, we used them to transform cells stripped of their cell walls (23, 24).

Fig. 4.
Fibers of CPEB-Q-M are sufficient for templating self-perpetuating change in CPEB activity. (A) Agitated CPEB-Q-M forms SDS-resistant amyloid polymers over time. Transmission electron micrographs show fibers and aggregates of CPEB-Q-M (B) and CPEB-Q ( ...

Blue and white cells transformed with the control plasmid (Fig. 4 D1 and D2) or with CPEB-Q-M in its soluble form (Fig. 4 D3 and D4) maintained their initial phenotypes: white cells never gave rise to blue, blue never gave rise to white. Complementary results were obtained with CPRG (Fig. 4 E1 and E2). However, blue cells did sometimes shift to a different shade of blue. Thus, although the stress of the protein transformation protocol did not induce or cure [CPEB+], it could cause cells to shift strain types (Fig. 4 D2 and E2).

In contrast, when CPEB-Q or CPEB-Q-M was introduced in the amyloid form, many white cells switched to the blue (Fig. 4D5). Importantly, blue cells transformed with the same preparations of CPEB-Q and CPEB-Q-M never gave rise to white. Again, complementary results were obtained with CPRG (Fig. 4E3). Similar results were obtained with several independently assembled fiber preparations, except that the efficiency of transformation varied from 30% to 90%. Transformation efficiencies were somewhat higher with CPEB-Q-M than with CPEB-Q.

We attempted to produce sufficient quantities of pure, strain-specific [CPEB+] templates for protein transformation using cell lysates to seed polymerization. Unfortunately, the off-pathway aggregation that takes over in nonagitated assembly reactions (Fig. 3) prevented us from generating sufficient material. In the more efficient rotated reactions, spontaneous assembly took over, again preventing strain-specific template amplification. However, the amyloids produced by spontaneous assembly of both CPEB-Q and CPEB-Q-M gave rise to the full range of blue [CPEB+] colors (Fig. 4D5 and E3).

These findings were confirmed to reflect different β-gal activities using the CPRG assay. Mating experiments confirmed the dominance of the transformation-acquired [CPEB+] state (Fig. S4A). Fiber-transformed cells remained blue, and retained their initial diverse color intensities, after continuous restreaking over the course of a full year (Fig. S4B). Thus, the diversity of [CPEB+] strain types are based on a protein-only prion mechanism.


An intriguing concept emerging in recent years is that cells of both lower and higher organisms can harness prion-based conformational changes to initiate beneficial switches in phenotype with the durability that is normally associated with replicating nucleic acids (2). We demonstrate that protein-only conformational changes in neuronal Aplysia CPEB suffice to generate not just one but multiple stable self-perpetuating CPEB activities. As demonstrated previously with yeast prions, amyloid conformers transmit and encode these [CPEB+] prion states (3, 2124). Unlike most yeast prions, however, it is the prion form of CPEB that is the active form.

How is it possible for CPEB to function in an assembled amyloid structure? One factor is the modularity of prion domains, which do not propagate their amyloid conformations into adjacent globular domains. In the fusion with CPEB-Q-M, EGFP retains its fold, remaining fluorescent when the prion domain enters the amyloid state. Sup35 and other yeast prion domains behave in the same manner. Indeed, electron microscopy of assembled full-length Sup35 reveals that the C-terminal translation domain forms an orderly array outside of the amyloid fiber core (32) (Fig. S5A). Full-length CPEB almost certainly does the same.

However, how could CPEB actually become more active in the prion state? There are at least two, nonexclusive possibilities. First, in its nonamyloid form the prion domain might inhibit the activity of CPEB's RNA binding domain. Assembly would sequester the inhibitor, freeing the C-terminal domain for function. Second, polymerization of CPEB would strongly increase its local concentration, allowing it to more efficiently recruit other factors involved in translational activation, and perhaps even to scaffold their assembly (Fig. S5B).

In the yeast system, prion-based conformational switches create heritable and self-perpetuating epigenetic elements. CPEB's prion states confer these same epigenetic properties in this heterologous system. Neurons are nondividing cells that cannot manifest such behaviors. However, the prion conformational switch is a biochemical type of memory that might be exploited for maintaining the structure of newly formed synapses, the essence of neuronal memory. It has long been known that CPEB activity plays a vital part in long-term facilitation in the neuron (15). In addition to promoting long-term maintenance of the active state, the assembly of CPEB into prion polymers would have an additional function at the synapse. Of the hundreds to thousands of connections that neurons make with other neurons, the essential character of memory is the regulation of individual synapses in particular circuitries (33, 34). As previously noted, the nondiffusible nature of CPEB prion assemblies would keep the active form of CPEB local to individual synapses, providing a maintenance function for the local synaptic mark and establishing long-term memory (13).

Like many of the well-characterized yeast prion proteins, CPEB has an intrinsic capacity to exist in several different fine-tuned activity states, or strains, which are remarkably stable and self-perpetuating. As is the case for strains of bona fide yeast prions, CPEB strains template soluble protein with different efficiencies. It is tempting to speculate that this capacity for finely tuned translational activity, with its associated differences in templating efficiencies, might be used by neurons to create synapses with different strengths and perhaps different durabilities.

In the heterologous yeast system, the appearance of CPEB strains had a stochastic character. However, [CPEB+] appears much more frequently than is typical of yeast prions. That is, this protein has an unusually high propensity to acquire a prion fold. Presumably, a factor that negatively regulates this fold in neurons is missing in yeast. Indeed, given the importance of synaptic memory, it seems certain that CPEB activity states will prove to be tightly regulated by multiple factors in neurons. (Indeed, even in yeast we found that the metabolic state of the cell influenced the frequency of the prion's appearance.) However, once these different states are established, by whatever neuronal factors control them, our findings suggest distinct activity states might be locally maintained by the intrinsic capacity of CPEB to self-perpetuate them. Yeast cells even provide a clue that these states, stable as they are, are subject to modulation. Although CPEB strains were normally very durable, switches between strain types occurred when cells were subject to the stresses of protein transformation. Therefore, the properties of CPEB strains could provide a local, rather than global, mechanism for synaptic homeostasis (35).

In CPEB's natural neuronal regulatory environment it would have been very difficult to unambiguously establish CPEB's intrinsic capacity for acquiring distinct, finely tuned activity states. The absence of neuronal conformation modulators in yeast now provides an ideal system to investigate neuron-specific modulators of CPEB functional status, by introducing them into CPEB-expressing yeast cells.

In addition to serving as a living test tube to investigate Aplysia neuronal CPEB activity, it is notable that yeast has a functional homolog of CPEB. This homolog, Hrp1, is part of the yeast polyadenylation machinery (19). Hrp1 also meets many of the experimental criteria for a yeast prion described in a comprehensive screen for new prion proteins (3). Indeed, several proteins of the polyadenylation complex (including Hrp1) carry domains enriched in glutamines and asparagines that resemble prion domains. Through its potential interaction with other putative prions in the polyadenylation machinery CPEB/Hrp1 may be part of an ancient prion-based mechanism of regulating mRNA activities in eukaryotic cells. Many newly identified yeast proteins with prion domains (3) interact, as does neuronal CPEB, with nucleic acid, either as RNA binding proteins (e.g., Pub1, Puf2) or transcription factors (e.g., Mot3, Swi1, Cyc8, Sfp1), which positions them, as well, to act as self-perpetuating regulators of gene expression. Thus, prion proteins may form a new system for durable epigenetic regulation of diverse cellular functions operating in a wide variety of biological contexts.

Materials and Methods

Constructs, Yeast Strains, and Media.

Aplysia CPEB was codon-optimized for expression in yeast (BioBasic, Inc.) and introduced into the Gateway entry vector pENTR/d-TOPO. Gateway LR reactions with 413GPD-ccdB, 413GPD-ccdB-EGFP, or 416GPD-ccdB-EGFP destination vectors produced GPD-ApCPEB and GPD-ApCPEB-EGFP (HIS+ or URA+). The N domain of pAED4-ScNM-his7 (yA7111) was replaced by codon-optimized CPEB-Q to produce his6-tagged CPEB-Q-M, and introduced into pENTR/d-TOPO. Gateway LR reactions with 426GAL-ccdB-EGFP produced GAL-CPEB-Q-M-EGFP. The βgal-CPE reporter, his6-tagged CPEB-Q constructs and noncodon-optimized CPEB were kindly provided by K. Si (Stowers Institute, Kansas City, MO) (16). CPEB-expressing yeast strains w303 MAT a or α, ADE+ (y4436 and y4437), BY4741 (Euroscarf) for fiber transformation and A3464 for cytoduction were grown on standard synthetic media lacking particular amino acids/bases, with d-glucose or d-galactose as a carbon source; details in SI Materials and Methods.

X-Gal and CPRG Assays.

X-Gal assays on plates were performed as described (16). For CPRG assays, overnight cultures were grown (starting OD600 0.2) in YPD buffer for 1h and an equal volume of CPRG assay buffer [100 mM hepes, pH 7.25, 150 mM NaCl, 5 mM l-aspartate, 1% BSA, 0.05% Tween, 0.5% SDS, 1.2 mM CPRG (884308; Roche)] was added.

Light and Electron Microscopy.

Overnight cell cultures with GPD-CPEB (CEN), βgal-CPE, and GAL-CPEB-Q-M-EGFP (2 μ) plasmids were grown (starting OD600 0.2) in galactose without uracil, histidine, and leucine to midlog phase and examined with an Axioplan microscope with a 100× objective (Zeiss). Photoshop was used for linear adjustments of brightness and contrast. For TEM, 5 μL of protein solution was applied to 200 mesh carbon-coated copper grids, stained with uranyl acetate and imaged as detailed in SI Materials and Methods.

Protein Preparation, Assembly, and Analysis.

SDD-AGE analysis of whole cell lysates or in vitro assembly reactions was performed as described (3, 28, 29). His6-tagged CPEB-Q-M and CPEB-Q proteins were purified under denaturing conditions with Ni+2-Agarose columns as described in SI Materials and Methods. CPEB-Q-M was used to test the seeding efficiency of various whole cell lysates in assembly buffer (0.2 M GdnHCl, 250 mM NaCl, 5 mM NaH2PO4, pH 7.5, 5 μg/mL aprotinin and leupeptin, 5 mM EDTA, 5 mM TCEP) for the indicated times using SDD-AGE and visualized with anti-his6 antibody (Invitrogen). For spontaneous assembly, purified his6-tagged CPEB-Q-M or CPEB-Q proteins were diluted into assembly buffer (250 mM NaCl, 5 mM NaH2PO4, pH 7.5, 5 mM EDTA, 5 mM TCEP) to a final concentration of 2 μM in 0.5 M or 1 M GdnHCl. Samples were rotated and 20-μL aliquots were removed at indicated times for analysis with SDD-AGE.

For fiber transformation, amyloid fibers of CPEB-Q-M and CPEB-Q were sonicated and transformed into a white or blue BY4741 strain expressing CPEB and carrying the βgal-CPE reporter gene using methodology previously described (23).


We thank K. Allendoerfer, K. Matlack, R. Halfmann, J. Goodman, J. Dong, L. Pepper, and D. Jarosz for critical reading of the manuscript and Thomas DiCesare for designing Fig. S5. We are especially grateful for the help and advice of Kausik Si and Eric Kandel. This work was supported by the G. Harold and Leila Y. Mathers Foundation (S.L.) and National Research Service Award Fellowship AGO30269 (to S.U.H.).


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019368108/-/DCSupplemental.


1. Aguzzi A, Baumann F, Bremer J. The prion's elusive reason for being. Annu Rev Neurosci. 2008;31:439–477. [PubMed]
2. Halfmann R, Alberti S, Lindquist S. Prions, protein homeostasis, and phenotypic diversity. Trends Cell Biol. 2010;20:125–133. [PMC free article] [PubMed]
3. Alberti S, Halfmann R, King O, Kapila A, Lindquist S. A systematic survey identifies prions and illuminates sequence features of prionogenic proteins. Cell. 2009;137:146–158. [PMC free article] [PubMed]
4. Shorter J, Lindquist S. Prions as adaptive conduits of memory and inheritance. Nat Rev Genet. 2005;6:435–450. [PubMed]
5. True HL, Berlin I, Lindquist SL. Epigenetic regulation of translation reveals hidden genetic variation to produce complex traits. Nature. 2004;431:184–187. [PubMed]
6. Eaglestone SS, Cox BS, Tuite MF. Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism. EMBO J. 1999;18:1974–1981. [PMC free article] [PubMed]
7. Treusch S, Cyr DM, Lindquist S. Amyloid deposits: Protection against toxic protein species? Cell Cycle. 2009;8:1668–1674. [PubMed]
8. Fowler DM, Koulov AV, Balch WE, Kelly JW. Functional amyloid—From bacteria to humans. Trends Biochem Sci. 2007;32:217–224. [PubMed]
9. Barnhart MM, Chapman MR. Curli biogenesis and function. Annu Rev Microbiol. 2006;60:131–147. [PMC free article] [PubMed]
10. Fowler DM, et al. Functional amyloid formation within mammalian tissue. PLoS Biol. 2006;4:e6. [PMC free article] [PubMed]
11. Maji SK, et al. Functional amyloids as natural storage of peptide hormones in pituitary secretory granules. Science. 2009;325:328–332. [PMC free article] [PubMed]
12. Tyedmers J, Madariaga ML, Lindquist S. Prion switching in response to environmental stress. PLoS Biol. 2008;6:e294. [PMC free article] [PubMed]
13. Bailey CH, Kandel ER, Si K. The persistence of long-term memory: A molecular approach to self-sustaining changes in learning-induced synaptic growth. Neuron. 2004;44:49–57. [PubMed]
14. Miniaci MC, et al. Sustained CPEB-dependent local protein synthesis is required to stabilize synaptic growth for persistence of long-term facilitation in Aplysia. Neuron. 2008;59:1024–1036. [PMC free article] [PubMed]
15. Si K, Choi YB, White-Grindley E, Majumdar A, Kandel ER. Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation. Cell. 2010;140:421–435. [PubMed]
16. Si K, et al. A neuronal isoform of CPEB regulates local protein synthesis and stabilizes synapse-specific long-term facilitation in aplysia. Cell. 2003;115:893–904. [PubMed]
17. Si K, Lindquist S, Kandel ER. A neuronal isoform of the aplysia CPEB has prion-like properties. Cell. 2003;115:879–891. [PubMed]
18. Richter JD. CPEB: A life in translation. Trends Biochem Sci. 2007;32:279–285. [PubMed]
19. Mandel CR, Bai Y, Tong L. Protein factors in pre-mRNA 3′-end processing. Cell Mol Life Sci. 2008;65:1099–1122. [PMC free article] [PubMed]
20. Keleman K, Krüttner S, Alenius M, Dickson BJ. Function of the Drosophila CPEB protein Orb2 in long-term courtship memory. Nat Neurosci. 2007;10:1587–1593. [PubMed]
21. Wang F, Wang X, Yuan CG, Ma J. Generating a prion with bacterially expressed recombinant prion protein. Science. 2010;327:1132–1135. [PMC free article] [PubMed]
22. Du Z, Park KW, Yu H, Fan Q, Li L. Newly identified prion linked to the chromatin-remodeling factor Swi1 in Saccharomyces cerevisiae. Nat Genet. 2008;40:460–465. [PMC free article] [PubMed]
23. Tanaka M, Chien P, Naber N, Cooke R, Weissman JS. Conformational variations in an infectious protein determine prion strain differences. Nature. 2004;428:323–328. [PubMed]
24. King CY, Diaz-Avalos R. Protein-only transmission of three yeast prion strains. Nature. 2004;428:319–323. [PubMed]
25. Wickner RB, Edskes HK, Shewmaker F. How to find a prion: [URE3], [PSI+] and [beta] Methods. 2006;39:3–8. [PubMed]
26. Li L, Lindquist S. Creating a protein-based element of inheritance. Science. 2000;287:661–664. [PubMed]
27. Liu JJ, Sondheimer N, Lindquist SL. Changes in the middle region of Sup35 profoundly alter the nature of epigenetic inheritance for the yeast prion [PSI+] Proc Natl Acad Sci USA. 2002;99(Suppl 4):16446–16453. [PMC free article] [PubMed]
28. Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD, Kushnirov VV. Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104. J Biol Chem. 2003;278:49636–49643. [PubMed]
29. Tyedmers J, et al. Prion induction involves an ancient system for the sequestration of aggregated proteins and heritable changes in prion fragmentation. Proc Natl Acad Sci USA. 2010;107:8633–8638. [PMC free article] [PubMed]
30. Douglas PM, et al. Chaperone-dependent amyloid assembly protects cells from prion toxicity. Proc Natl Acad Sci USA. 2008;105:7206–7211. [PMC free article] [PubMed]
31. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science. 1982;216:136–144. [PubMed]
32. Glover JR, et al. Self-seeded fibers formed by Sup35, the protein determinant of [PSI+], a heritable prion-like factor of S. cerevisiae. Cell. 1997;89:811–819. [PubMed]
33. Frey U, Morris RG. Synaptic tagging and long-term potentiation. Nature. 1997;385:533–536. [PubMed]
34. Martin KC, et al. Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: A function for local protein synthesis in memory storage. Cell. 1997;91:927–938. [PubMed]
35. Rabinowitch I, Segev I. Two opposing plasticity mechanisms pulling a single synapse. Trends Neurosci. 2008;31:377–383. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...