Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation*

The cellular prion protein (PrP^C)² underlies a group of fatal neurodegenerative diseases through its conversion into self-perpetuating and neurotoxic forms (1–4). Despite a large amount of evidence supporting a role in survival/death and growth/differentiation cell decisions, the physiological function of PrP^C and its involvement in disease remain elusive (5–8). A crucial limiting factor for PrP^C functional determination is its molecular diversity. Although PrP^C is mainly thought of as a glycoprotein attached to the cell surface by a glycosylphosphatidylinositol anchor, PrP^C is actually synthesized as a family of four members: the membrane anchored glycoprotein (^SecPrP), two transmembrane forms with opposite topologies (^NtmPrP and ^CtmPrP), and a soluble form (^CytPrP) (3, 9–12).

Of these different members, ^CytPrP, accounts for a minor intracellular subset of PrP^C that has attracted much attention because its accumulation sensitizes cells to death (13–15). Initially, ^CytPrP was thought to be formed by misfolded chains that retrotranslocated through the endoplasmic reticulum-associated protein degradation-proteasome pathway (16, 17). However, it was later shown that ^CytPrP is constitutively populated by nascent chains that spill into the cytosol due to inefficient N-terminal signaling (15, 18). Regarding the role of ^CytPrP, most knowledge has been provided by models consisting of mutant polypeptide chains that are inappropriately expressed and folded in the cytosol. These PrP(23–230) chains exhibit a widespread intracellular distribution (19–21) and an alleged role that varies from cytotoxic (14, 20) to innocuous or even protective (19, 22, 23). These contradictions call into question the fidelity with which such models can describe ^CytPrP.

The finding that information for ^CytPrP synthesis is contained in its N-terminal signal sequence (15) prompted us to decipher this code and use it as a tool to isolate its synthesis from that of the major forms and inspect its function. We have found that ^CytPrP is indeed a novel PrP isoform that accesses the nucleus and interferes with cell growth. These results provide new insights on PrP diversity and its role in health and disease.

EXPERIMENTAL PROCEDURES

Plasmid Construction and Recombinant Standard Production—The plasmid pcDNA4-HaPrP, kindly provided by Dr. R. S. Hegde, was first mutated to introduce the six nucleotides from the 5′-untranslated region adjacent to the initial ATG to preserve the wild type Kozak sequence. The HuPrP open reading frame was cloned into pcDNA3.1 at BamHI/EcoRI sites preserving the corresponding wild type Kozak region. Wild type constructs were used as templates to generate different mutants (see Table 1 and Fig. 1) by using the Quikchange protocols (Stratagene). The integrity of each construct was verified by sequencing. Recombinant PrP chains of 1–254, 15–231, 15–254, and 23–231 were produced from the corresponding pET11a plasmids in Escherichia coli BL21(DE3) and used as inclusion bodies denatured extracts as described previously (5).

Transcription, Translation, and Translocation Assays—All plasmids were enzymatically linearized (pcDNA4.1-HaPrP plasmids with ApaI and pcDNA3.1-HuPrP constructs with SacII) and then transcribed with the T7 CapScribe kit (Promega). After integrity verification, the transcribed mRNAs were translated at 80 μg/ml final concentration using the 50% (v/v) nuclease-treated rabbit reticulocyte lysate system (Promega) and Redivue™ l-[³⁵S]methionine (Amersham Biosciences), as indicated by the manufacturer. For translation-translocation assays, the reaction mixture was enriched in 15% (v/v) canine pancreatic rough microsomal membranes (Ref. 24 and references therein). Isolation of the fraction of sealed microsomes from the reaction mixtures was performed by discontinuous sucrose gradient ultracentrifugation as described previously (24). For protease protection analysis, the total reaction mixtures and their sealed microsomal fractions were incubated for 1 h at 4 °C with 0.1 mg/ml proteinase K (Roche Diagnostics) both in the absence and in the presence of 0.5% Triton X-100. The reaction was stopped with 5 mm phenylmethylsulfonyl fluoride. The ³⁵S-labeled reaction products were immunoprecipitated with αPrP 3F4 monoclonal antibody (Signet Laboratories), resolved on Tris-Tricine 16.5% SDS-PAGE gels, and visualized using a phosphorimaging device (Fuji FLA-3000). Enzymatic deglycosylation was performed by incubating the immunoprecipitated samples with PNGase F (New England Biolabs) according to the manufacturer's instructions.

Cell Culture, Transfections, and Treatments—CHO and COS-7 cells were grown and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 10 IU/ml penicillin, and 10 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37 °C. Transfection of cells with the different plasmids was performed with TransIT-LT1 (Mirus) following the manufacturer's indications. After 48 h, cells were processed for analysis or used for bulk selection of stable transfectants. For proteasome impairment experiments, 24 h after transfection, cells were treated in the absence or presence of 5 μm MG132 for 18 h (15). Alternatively, after 4 h, the medium was changed, and the incubation was continued for another 14 h (15). After PBS washes, cells were harvested and analyzed for protein aggregation (see below).

Cell Lysates, Brain Homogenates, and Fractionations—Denatured cell lysates were prepared at about 15 mg/ml protein concentration in 62.5 mm Tris-HCl, pH 6.8, containing 4% SDS (w/v) and 25% glycerol (w/v), boiled for 10 min, and then cleared by centrifugation at 15,000 × g for 20 min. Hamster brains were obtained from the Animal Facility of the Instituto de Investigaciones Biomédicas “Alberto Sols” Universidad Autónoma de Madrid (UAM)-CSIC. Human cortex control samples were obtained from the Institute of Neuropathology and University of Barcelona/Clinic Hospital brain banks following the guidelines of the local ethics committees. Tissue homogenates at 10% (w/v) were prepared in PBS, pH 7.5, containing 0.25 m sucrose, 1.5 mm sodium orthovanadate, 5 mm EDTA, and the EDTA-free Complete protein inhibitor mixture (Roche Diagnostics), aliquoted, and kept at –80 °C. Fractionation of cell and tissue homogenates into nuclear and postnuclear fractions was performed using the Pure Prep nuclei isolation kit (Sigma) following the manufacturer's indications. The final step including PIPLC digestion was introduced to ensure the removal of contaminant raft-resident PrP^C.

Protein Aggregation Assays—Analysis of PrP aggregation upon proteasome inhibition was performed with minor modifications to published methods (13, 15–17). Cells were lysed in cold EZ-lysis buffer and then separated into pellet (nuclear) and supernatant (postnuclear) fractions by a 500 × g centrifugation for 10 min at 4 °C. The pellets were washed twice with EZ-lysis buffer for isolation of the nuclear fractions. The postnuclear supernatants were supplemented with 0.5% Triton X-100 and 0.5% deoxycholate, dispersed by extensive pipetting, and then centrifuged for 10 min at 13,000 × g at 4 °C. Proteins in the supernatant were precipitated with cold 15% trichloroacetic acid. All protein pellets were resuspended in 0.1 m Tris-HCl, pH 8.0, 1% SDS, and equal aliquots of each fraction were analyzed by SDS-PAGE and immunoblotting.

Immunoprecipitation and Western Blotting—Samples were lysed in 62.5 mm Tris-HCl, pH 6.8, containing 4% (w/v) SDS, and 25% (w/v) glycerol. After a 10-min spin at 10,000 × g, the supernatants were diluted 1:40 with PBS, pH 7.4, containing 0.1% sodium deoxycholate (Calbiochem), 1% Nonidet P-40 (Sigma-Aldrich), 1.5 mm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride. Samples were incubated for 1 h with protein A/G-Sepharose (Amersham Biosciences). After a 5-min centrifugation at 200 × g, the supernatants were incubated with either 3F4 or SP α-PrP antibodies at 4 °C, and the resulting immunocomplexes were captured with protein A/G-Sepharose and released using Laemmli buffer. Proteins (∼50 μg/lane) were separated by electrophoresis on 13% SDS-polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (150 V, 1 h). The membrane-bound proteins were probed with the primary antibody followed by mouse TrueBlot horseradish peroxidase-conjugated α-mouse IgG (1:1000, eBioscience), goat α-mouse horseradish peroxidase-conjugated IgG (1:3000, Sigma), or goat α-rabbit horseradish peroxidase-conjugated IgG (1:8000, Chemicon) and then developed with chemiluminescent Chemicon reagents. Data acquisition and analysis were carried out using the Bio-Rad ChemiDoc equipment. The following primary antibodies and dilutions were used: αPrP 3F4 (1:5000, Signet Laboratories), αPrP SP (1:1000, provided by D. Harris and raised against PrP N-terminal signal peptide), α-YFP (1:3000, α-green fluorescent protein AbCam), α-PDI (1:1000, AbCam), α-β-coat protein (1:1000, AbCam), α-histone H3 (1:2000, AbCam), α-ubiquitin P4D1 (1:200, Santa Cruz Biotechnology), α-SUMO-1 D-11 (1:200, Santa Cruz Biotechnology), and β-actin (1:5000, Sigma-Aldrich).

Confocal Fluorescence Microscopy—Cells were plated onto glass coverslips, allowed to attach for 24 h, and then transfected for 48 h. Cells were fixed with 4% paraformaldehyde in PBS containing 5% sucrose for 10 min at room temperature and washed three times with PBS. Cells were permeabilized and blocked in PBS containing 0.5% saponin, 0.1% Triton, and 2% bovine serum albumin for 10 min at room temperature. Cells were incubated with αPrP 3F4 (1:600) and with α-PDI (1:600) for 1 h at room temperature. After three washes with blocking buffer, samples were incubated with Alexa Fluor-647-conjugated goat α-mouse IgG (1:800), Alexa Fluor-488-conjugated α-rabbit IgG (1:800), and Hoechst 33342 (10 μg/ml) in blocking solution for 30 min at room temperature. After washing, the coverslips were mounted on glass slides with ProLong Gold antifade reagent (Molecular Probes). Images were captured with a confocal microscope (Leica TCS-SP-AOBS-UV) using the UV and argon lasers at 20 milliwatt for excitations at 364 nm (Hoescht) and 488 nm (Alexa Fluor-488), respectively, and the 633-nm line of the He-Ne laser at 10 milliwatt for excitation at 647 nm. Image analysis was performed using Leica confocal software.

Cell Proliferation Assays and Cell Cycle Analysis—For cell growth analysis, cells were co-transfected with pEYFP (Clontech) and the plasmid coding wild type HaPrP or its mutants. After 48 h of transfection, cells were synchronized in G₀/G₁ by serum deprivation for 18 h and then released by serum supplementation for 6 h. Cell proliferation was analyzed in the 96-well format using the bromodeoxyuridine cell proliferation kit (Calbiochem) and a MR500 microplate reader (Dynatech). Bromodeoxyuridine labeling was performed for 6 h during the 10% fetal bovine serum stimulation period. Cell cycle profiles were determined by flow cytometry using the standard measurement of DNA content with propidium iodide in a BD FACSCalibur cytometer (BD Biosciences). In this case, YFP-positive cells were selected by cell sorting before propidium iodide labeling. Data were compared by one- or two-way analysis of variance with Bonferroni's post-test analysis using GraphPad Prism v 4.0.

RESULTS

N-terminal Signal Peptides of PrP Contain a Dual Methionine Motif—N-terminal signal peptides display a tripartite organization into n-, h-, and c-regions, with the hydrophobic central region (h-region) essential for co-translation membrane integration and translocation process. The signal sequences of PrP from different species can be classified into three groups on the basis of the number of Met residues and their position with respect to regional boundaries (Fig. 1). Group I, represented by the rodent sequences, contains two Met residues at positions 1 and 15; the second position is in the N-terminal side of the c-region. In Group II, represented by the human sequence, the two Met residues are at positions 1 and 8. In this case, the second Met constitutes the N terminus of the h-region. On the contrary, Group III, which is represented by the mink sequence, lacks the second Met residue. When converted into their cognate mRNA sequences, the Met residues of the signal sequences become AUG codons that could behave as translation initiation sites. We also identified two in-frame triplets (CUG and GUG coding HaPrP L9 and V13) that could sustain translation initiation by means of a single base difference. These non-AUG codons are conserved in all species. If used, any of these codons could yield nascent chains with different cellular fates.

The MM Motif Allows a Dual Translation Start and the Existence of PrP Isoforms—To test whether the downstream AUG codons found in the signal sequence regions of PrP mRNA in Group I and Group II could sustain translation initiation, we generated a series of point mutations in both the HaPrP and the HuPrP open reading frames (Figs. 1 and 2 and Table 1). These mutations consisted of the insertion of a C or a G at various positions causing a +1 shift in the reading frame (11C12, 13G14, 16C17), as well as an Met-to-Ser substitution (ATG-to-TTC). The reading frameshift mutations allow the study of both non-AUG and AUG start sites, whereas the Met-to-Ser substitutions permit the evaluation of the role of a specific Met residue. It should be noted that frameshift mutations allow translation initiation at either start site, but only the product produced from the start site downstream from the insertion will proceed to the wild type (WT) stop codon and will produce chains retaining the 3F4 PrP epitope.

The results of the translation of the mRNAs coding for WT and mutant HaPrP and HuPrP using reticulocyte lysates followed by immunoprecipitation with 3F4 are shown in Fig. 2A. In agreement with previous reports, wild type HaPrP mRNA was translated into a major polypeptide chain of about 26 kDa. Under the same conditions, translation of the mRNA coding HaPrP(M1S), in which the canonical AUG is functionally impaired, and for the reading frameshift mutants HaPrP(11C12) and HaPrP(13G14) led to the production of a single product of similar mass (26 kDa) but with reduced intensity (about 10–15% of that of the WT). On the contrary, translation of the mRNAs coding HaPrP(16C17) and HaPrP(M1S,M15S) resulted in the absence of any detectable signal. These results support the idea that HaPrP mRNA contains a minor translation initiation site and that this site is located at codon 15, the AUG triplet coding for Met-15. It should be noted that the chains translated from Met-1 and Met-15 could not be easily differentiated by electrophoresis, probably as a result of the balance between the differences in size and hydrophobicity of the chains (25).

The translation of WT HuPrP mRNA yielded a band corresponding to a polypeptide chain of about 27 kDa (Fig. 2A). This band was detected using the mRNAs of the HuPrP(M1S) and HuPrP(5C6) mutants but with less intensity. On the contrary, this band was not observed using the mRNAs of the HuPrP(11C12), HuPrP(13C14), HuPrP(16C17), and HuPrP(M1S,M8S) mutants. These results show that HuPrP mRNA, as model for Group II, also contains a minor translation start site and that this site is located at codon 8, the AUG triplet coding for Met-8. Because the alternative translation start site of PrP signal sequences in groups I and II is due to the dual methionine motif, it follows that the sequences of Group III either lack this capacity or utilize a different process.

HaPrP(M15) and HuPrP(M8) Isoforms Account for de Novo Synthesized ^CytPrP—To unambiguously establish the relationship between HaPrP(M15) and HuPrP(M8) and de novo synthesized ^CytPrP, we studied their behavior in cell-free biosynthesis assays (Fig. 2, B and C). Fig. 2B shows that in contrast to the WT mRNAs, the products of mRNAs coding HaPrP(M1S) and HuPrP(M1S) mutants and translated in the presence of microsomal membranes consisted of a single band of ∼26 kDa that remained unchanged after PNGase F digestion. Comparison of the bands after deglycosilation, in particular of HuPrP chains, suggests that HuPrP(M1S) migrates similarly to an unprocessed full-length chain (see below) (26). The unglycosylated pattern agrees with a cytosolic location for the HaPrP(M15) and HuPrP(M8) C-terminal domains. Furthermore, external addition of proteinase K to both the total reaction mixture and its sealed microsome fraction (no signal was detected in this fraction even using a 10× overload as compared with the WT) resulted in complete degradation of the ∼26-kDa chains translated from the HaPrP(M15) and HuPrP(M8) mRNAs (Fig. 2C). In contrast, the product translated from WT mRNAs under similar conditions showed protected fragments corresponding to translocated and integrated PrP chains (3). Taken together, these results suggest that HaPrP(M15) and HuPrP(M8) chains segregate outside the secretory route under a proteinase K-sensitive conformation as described for ^CytPrP.

To determine whether the synthesis of these isoforms takes place in cellular contexts, we proceeded with transient transfection experiments using CHO and COS-7 cells, which have undetectable levels of endogenous PrP expression. In this case, the study was restricted to the HaPrP sequences for biosafety reasons and was performed as co-transfection with pEYFP to use YFP expression as an internal control. Plasmids encoding HaPrP WT and HaPrP(M1SM15S) were used as positive and negative controls for PrP^C expression, respectively, whereas those encoding HaPrP(11C12) and HaPrP(M1S) were employed to assess the functionality of M15 as start site. Fig. 3A shows that HaPrP(M15) was indeed synthesized by cells based on the presence of a 26-kDa band recognized by αPrP 3F4 in the lysates of HaPrP(11C12) and HaPrP(M1S) transfectants. Importantly, the 26-kDa band was also recognized by α-SP, an antibody raised against the C-terminal region of the signal sequence (11). In cell lysates, HaPrP(M15) retained the C-terminal hydrophobic segment according to electrophoretic mobility determinations using a panel of recombinant PrP chains consisting of the full unprocessed chain (1–254 sequence), the fully processed chain (23–231 sequence), and N-terminally shortened chains either containing (15–254) or lacking (15–231) the C-terminal hydrophobic segment (Fig. 3B).

To corroborate that the HaPrP(M15) synthesized in cells behaves as ^CytPrP, we studied its glycosylation state as well as its capacity to form insoluble aggregates upon proteasome impairment (13, 15–17). Fig. 3C shows that in contrast to WT HaPrP, 26-kDa HaPrP(M1S) remained unchanged upon PNGase F digestion as expected for ^CytPrP. Moreover, both transient and irreversible inhibition of the proteasome with 5 μm MG132 promoted the formation in the cytosol of insoluble HaPrP(M1S) aggregates (Fig. 3D). Both the absence of glycosylation and the capacity to form cytosolic insoluble aggregates confirms that HaPrP(M15) behaves as ^CytPrP in a cellular context.

HaPrP(M15) and HuPrP(M8) Are Found in Nuclei Isolated from Cells and Normal Brain Homogenates and Are Sumoylated—To elucidate the properties of these isoforms, we first studied their subcellular location using confocal microscopy. Unless stated, HaPrP(M15) was expressed from the HaPrP(Δ14) construct for easier detection. Indirect immunofluorescence stainings showed that at 48 h after transfection, HaPrP(M15) was localized largely to the nuclei of cells (Fig. 4A). The distribution pattern agreed with the diffuse nucleoplasmic location observed for several studied ^CytPrP models (19, 27) and differed from the intranuclear granules observed in neuronal cells expressing bovine PrP^C (28). The nuclear localization was then confirmed by subcellular fractionation of cell homogenates. Fig. 4B shows that about 70% of the expressed HaPrP(Δ14) was localized to the nuclear fraction, mainly as a 26-kDa chain but also as higher molecular weight species. Similar results were obtained using CHO and COS-7 cells.

To generalize the nuclear localization of HaPrP(M15), as well as to determine the origin of the high molecular weight bands, we purified the nuclei from normal hamster brain and human cortex homogenates and characterized the PrP contained therein. Before the analysis, the purified nuclei were dispersed in EZ-lysis buffer, digested with PIPLC, and then centrifuged at low speed. This process allows the release of the contaminant membrane-anchored forms (29). Fig. 5A shows that after removing raft-resident PrP^C, PrP was detected in the nuclei purified from hamster brain homogenates as two bands of 26 and 35 kDa that remained unchanged after enzymatic deglycosylation. Nuclear PrP in human cortex was also comprised of two major PNGase F-resistant bands of about 26 and 35 kDa. These bands were recognized by both 3F4 and SP, as expected from PrP chains bearing N-terminal shortened signal peptides (Fig. 5B). These data confirm the existence and nuclear distribution of isoforms produced by alternative translation in normal tissues.

The complexity of the bands suggests the occurrence of covalent modifications. Of the modifications that can occur in nuclear proteins and cause increases in size, activity-modifying sumoylation and degradation-targeting ubiquitinylation were studied. Fig. 5C shows that high molecular weight bands of PrP immunoprecipitated with 3F4 from denatured nuclei extracts of hamster brain homogenates were recognized by an anti-SUMO-1 antibody but not by anti-ubiquitin or anti-SUMO 2/3 antibodies. Inverse pull-down experiments with α-SUMO-1 confirmed 3F4 immunoreactivity. Because SUMO-1 conjugation involves the covalent attachment of a single 9.5-kDa chain, the observed band pattern can be explained to a large extent by considering the composition of a non-sumoylated chain (26 kDa) and a SUMO-1-conjugated form (35 kDa). In summary, PrP(M8/M15) appear to be a nuclear isoform that acts as substrate for SUMO-1 conjugation.

HaPrP(M15) Expression Abrogates Cell Proliferation—Trials to establish cell lines expressing HaPrP(M15) were unsuccessful despite the absence of a conclusive and reproducible cell death event. Stably transfected clones were selected, but they failed to grow. These growth alterations together with the nuclear distribution and involvement of reversible sumoylation prompted us to consider a possible antiproliferative activity.

Analysis of bromodeoxyuridine incorporation showed that HaPrP(M15) did indeed decrease cell growth as compared with HaPrP WT and the negative control HaPrP(M1SM15S) in both COS-7 and CHO cells (Fig. 6A). This effect was more pronounced and statistically significantly higher (p < 0.001) for the HaPrP(Δ14) mutant, which overexpresses the PrP isoform (Fig. 3A), than for the HaPrP(M1S) and HaPrP(11C12) mutants.

The cell cycle was then analyzed using a co-transfection approach. In this case, cells were co-transfected with pEYFP for separation of the positive transfectants by cell sorting before propidium iodide labeling. Fig. 6, B and C, shows that cells expressing HaPrP(M15) from both HaPrP(Δ14) and HaPrP(11C12) constructs exhibited a higher proportion of cells in the G₀/G₁ phase as compared with the mock control (transfection with HaPrP(M1SM15S)). These results indicate that HaPrP(M15) functions as a growth suppressor that delays the exit from G₁ phase.

DISCUSSION

In this study, we have shown that the minor member of the PrP^C family segregating outside the secretory route is generated by alternative initiation of translation. The presence of a second Met residue at the h-region boundary of the signal sequence determines the alternative translation initiation event. This process permits the synthesis of an isoform translated either from Met-15 in HaPrP or from Met-8 in HuPrP. This isoform represents a novel chain differing from the conventional mature form in the retention of the c-region of the N-terminal signal sequence and the full C-terminal hydrophobic region. These two segments could provide new functions as stability regulators or as sites of interaction for distinct ligands, among others.

The use of in-frame alternative translation start sites is a relatively common process by which proteins encoded by a single mRNA can acquire a multiplicity of sorting and function under environmental regulation (30–34). Although the relative proportion of PrP(M15) synthesis was about 12% both in the in vitro studies and in the cell systems used for transfection, it might be susceptible to such modulation. This is supported by the cell and regional dependence of ^CytPrP in normal rodent brains as well as its increased levels under endoplasmic reticulum stress (23, 35). As noted, the dual start site motif is missing in a group of highly conserved PrP sequences. In these sequences, the AUG triplet coding for Met-15 is found as either ACG or ACA, which code for Thr. Of these two codons, ACG can function as a non-AUG start site (36, 37). However, the function of the ACA triplet as a start is unclear; thus whether ACA-bearing species use the alternative translation mechanism remains to be established.

Isolating the synthesis of HaPrP(M15) from that of the major membrane-bound PrP forms allowed three major findings: nuclear localization, variable SUMO-1 conjugation, and anti-proliferative activity. The nuclear localization of this isoform might explain results of previous PrP studies describing rare nuclear localization, the presence of nuclear localization sequence, and the capacity of the chain to interact with nucleic acids and with chromatin (19, 28, 38).

SUMO-1 conjugation of the nuclear population of PrP suggests stringent regulation of activity and physiological relevance for this isoform. In general, sumoylation provides an on/off functional switch for protein interactions involved in processes such as transport, transcriptional silencing, genomic stabilization, and stress responses (39). SUMO-1-conjugated and free HaPrP(M15) chains might thus represent alternative functional states of the molecule. It is thus interesting to note that the degree of sumoylation in nuclei from brains was higher than that in cells. With the limitations imposed by the lack of sumoylation control, HaPrP(M15) expression might be involved in dysregulation of cellular growth resulting in G₀/G₁ phase arrest.

The antiproliferative function of HaPrP(M15) expands the physiological role of PrP^C. Because most cells withdraw from the cell cycle to differentiate during the G₁ phase, it is tempting to consider HaPrP(M15) as candidate for promotion of G₁ phase arrest required for cell differentiation in some developing tissues (7). On the other hand, the loss of HaPrP(M15) nuclear functionality might favor either cell transformation on depletion (8) or cell death on cytosolic accumulation (15). HaPrP(M15) can regulate the efficiency of prion accumulation, which decreases with cell division (40). The isolation of the synthesis of this isoform from that of other members of the PrP^C family suggests that each member of this family might have different physiological roles and that their aberrant cross-talk could also constitute a pathogenic mechanism.