A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons

(Ctm)PrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. In previous studies, we found that PrP molecules carrying mutations in the N-terminal signal peptide (L9R) and the transmembrane domain (3AV) were synthesized exclusively in the (Ctm)PrP form in transfected cell lines. To characterize the properties of (Ctm)PrP in a neuronal setting, we have utilized cerebellar granule neurons cultured from Tg(L9R-3AV) mice that developed a fatal neurodegenerative illness. We found that about half of the L9R-3AV PrP synthesized in these neurons represents (Ctm)PrP, with the rest being (Sec)PrP, the glycolipid anchored form that does not span the membrane. Both forms contained an uncleaved signal peptide, and they are differentially glycosylated. (Sec)PrP was localized on the surface of neuronal processes. Most surprisingly, (Ctm)PrP was concentrated in the Golgi apparatus, rather in the endoplasmic reticulum as it is in transfected cell lines. Our study is the first to analyze the properties of (Ctm)PrP in a neuronal context, and our results suggest new hypotheses about how this form may exert its neurotoxic effects.     

Cell surface accumulation of a truncated transmembrane prion protein in Gerstmann-Straussler-Scheinker disease P102L

A familial prion disorder with a proline to leucine substitution at residue 102 of the prion protein (PrP(102L)) is typically associated with protease-resistant PrP fragments (PrP(Sc)) in the brain parenchyma that are infectious to recipient animals. When modeled in transgenic mice, a fatal neurodegenerative disease develops, but, unlike the human counterpart, PrP(Sc) is lacking and transmission to recipient animals is questionable. Alternate mice expressing a single copy of PrP(102L) (mouse PrP(101L)) do not develop spontaneous disease, but show dramatic susceptibility to PrP(Sc) isolates from different species. To understand these discrepant results, we studied the biogenesis of human PrP(102L) in a cell model. Here, we report that cells expressing PrP(102L) show decreased expression of the normal 18-kDa fragment on the plasma membrane. Instead, a 20-kDa fragment, probably derived from transmembrane PrP ((Ctm)PrP), accumulates on the cell surface. Because the 20-kDa fragment includes an amyloidogenic region of PrP that is disrupted in the 18-kDa form, increased surface expression of 20-kDa fragment may enhance the susceptibility of these cells to PrP(Sc) infection by providing an optimal substrate, or by amplifying the neurotoxic signal of PrP(Sc). Thus, altered susceptibility of PrP(101L) mice to exogenous PrP(Sc) may be mediated by the 20-kDa (Ctm)PrP fragment, rather than PrP(102L) per se.     

A transmembrane form of the prion protein contains an uncleaved signal peptide and is retained in the endoplasmic Reticulum

Although there is considerable evidence that PrP(Sc) is the infectious form of the prion protein, it has recently been proposed that a transmembrane variant called (Ctm)PrP is the direct cause of prion-associated neurodegeneration. We report here, using a mutant form of PrP that is synthesized exclusively with the (Ctm)PrP topology, that (Ctm)PrP is retained in the endoplasmic reticulum and is degraded by the proteasome. We also demonstrate that (Ctm)PrP contains an uncleaved, N-terminal signal peptide as well as a C-terminal glycolipid anchor. These results provide insight into general mechanisms that control the topology of membrane proteins during their synthesis in the endoplasmic reticulum, and they also suggest possible cellular pathways by which (Ctm)PrP may cause disease.     

Most pathogenic mutations do not alter the membrane topology of the prion protein

The prion protein (PrP), a glycolipid-anchored membrane glycoprotein, contains a conserved hydrophobic sequence that can span the lipid bilayer in either direction, resulting in two transmembrane forms designated (Ntm)PrP and (Ctm)PrP. Previous studies have shown that the proportion of (Ctm)PrP is increased by mutations in the membrane-spanning segment, and it has been hypothesized that (Ctm)PrP represents a key intermediate in the pathway of prion-induced neurodegeneration. To further test this idea, we have surveyed a number of mutations associated with familial prion diseases to determine whether they alter the proportions of (Ntm)PrP and (Ctm)PrP produced in vitro, in transfected cells, and in transgenic mice. For the in vitro experiments, PrP mRNA was translated in the presence of murine thymoma microsomes which, in contrast to the canine pancreatic microsomes used in previous studies, are capable of efficient glycolipidation. We confirmed that mutations within or near the transmembrane domain enhance the formation of (Ctm)PrP, and we demonstrate for the first time that this species contains a C-terminal glycolipid anchor, thus exhibiting an unusual, dual mode of membrane attachment. However, we find that pathogenic mutations in other regions of the molecule have no effect on the amounts of (Ctm)PrP and (Ntm)PrP, arguing against the proposition that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases.     

Cotranslational partitioning of nascent prion protein into multiple populations at the translocation channel

The decisive events that direct a single polypeptide such as the prion protein (PrP) to be synthesized at the endoplasmic reticulum in both fully translocated and transmembrane forms are poorly understood. In this study, we demonstrate that the topological heterogeneity of PrP is determined cotranslationally, while at the translocation channel. By evaluating sequential intermediates during PrP topogenesis, we find that signal sequence-mediated initiation of translocation results in an interaction between nascent PrP and endoplasmic reticulum chaperones, committing the N terminus to the lumen. Synthesis of the transmembrane domain before completion of this step allows it to direct the generation of (Ctm)PrP, a transmembrane form with its N terminus in the cytosol. Thus, segregation of nascent PrP into different topological configurations is critically dependent on the precise timing of signal-mediated initiation of N-terminus translocation. Consequently, this step could be experimentally tuned to modify PrP topogenesis, including complete reversal of the elevated (Ctm)PrP caused by disease-associated mutations in the transmembrane domain. These results delineate the sequence of events involved in PrP biogenesis, explain the mechanism of action of (Ctm)PrP-favoring mutations associated with neurodegenerative disease, and more generally, reveal that translocation substrates can be cotranslationally partitioned into multiple populations at the translocon.     

Cell-specific metabolism and pathogenesis of transmembrane prion protein

The C-transmembrane form of prion protein ((Ctm)PrP) has been implicated in prion disease pathogenesis, but the factors underlying its biogenesis and cyotoxic potential remain unclear. Here we show that (Ctm)PrP interferes with cytokinesis in cell lines where it is transported to the plasma membrane. These cells fail to separate following cell division, assume a variety of shapes and sizes, and contain multiple nuclei, some of which are pyknotic. Furthermore, the synthesis and transport of (Ctm)PrP to the plasma membrane are modulated through a complex interaction between cis- and trans-acting factors and the endoplasmic reticulum translocation machinery. Thus, insertion of eight amino acids before or within the N region of the N signal peptide (N-SP) of PrP results in the exclusive synthesis of (Ctm)PrP regardless of the charge conferred to the N region. Subsequent processing and transport of (Ctm)PrP are modulated by specific amino acids in the N region of the N-SP and by the cell line of expression. Although the trigger for (Ctm)PrP upregulation in naturally occurring prion disorders remains elusive, these data highlight the underlying mechanisms of (Ctm)PrP biogenesis and neurotoxicity and reinforce the idea that (Ctm)PrP may serve as the proximate cause of neuronal death in certain prion disorders.     

Contact-induced structure transformation in transmembrane prion propagation

Based on recent experimental evidences of the transmission of prion diseases due to a particular transmembrane form (termed (Ctm)PrP), we propose a theoretical model for the molecular mechanism of such conformational diseases, in which a misfolded (Ctm)PrP induces a similar misfolding of another (Ctm)PrP. Computer simulations are performed to investigate the correlation between folding time and the concentration of misfolded PrP in various processes, including dimerization, trimerization, and cooperative dimerization. By comparing with the experimental correlation curve between incubation time and injected dose of scrapie prions, we conclude that cooperative dimerization may play an important role in the pathological mechanism of prion diseases.     

Cytosolic prion protein is the predominant anti-Bax prion protein form: exclusion of transmembrane and secreted prion protein forms in the anti-Bax function

Prion protein (PrP) prevents Bax-mediated cell death by inhibiting the initial Bax conformational change that converts cytosolic Bax into a pro-apoptotic protein. PrP is mostly a glycophosphatidylinositol-anchored cell surface protein but it is also retrotranslocated into cytosolic PrP (CyPrP) or can become a type 1 or type 2 transmembrane protein. To determine the form and subcellular location of the PrP that has anti-Bax function, we co-expressed various Syrian hamster PrP (SHaPrP) mutants that favour specific PrP topologies and subcellular localization with N-terminally green fluorescent protein tagged pro-apoptotic Bax (EGFP-Bax) in MCF-7 cells and primary human neurons. Mutants that generate both CyPrP and secreted PrP ((Sec)PrP) or only CyPrP have anti-Bax activity. Mutants that produce (Ctm)PrP or (Ntm)PrP lose the anti-Bax activity, despite their ability to also make (Sec)PrP. Transmembrane-generating mutants do not produce CyPrP and both normal and cognate mutant forms of CyPrP rescue against the loss of anti-Bax activity. (Sec)PrP-generating constructs also produce non-membrane attached (Sec)PrP. However, this form of PrP has minimal anti-Bax activity. We conclude that CyPrP is the predominant form of PrP with anti-Bax function. These results imply that the retrotranslocation of PrP encompasses a survival function and is not merely a pathway for the proteasomal degradation of misfolded protein.     

Mammalian Prion protein expression in yeast; a model for transmembrane insertion

The prion protein (PrP), a GPI-anchored glycoprotein, is inefficiently secreted by mammalian microsomes, 50% being found as transmembrane (TM) proteins with the central TM1 segment spanning the membrane. TM1 hydrophobicity is marginal for lateral membrane insertion, which is primarily driven by hydrophobic interaction between the ER translocon and substrates in transit. Most inserted TM1 has its N-terminus in the ER lumen (Ntm orientation), as expected for arrest of normal secretion. However, 20% is found in inverted Ctm orientation. These are minor species in vivo, presumably a consequence of efficient quality control. PrP mutations that increase TM1 hydrophobicity result in increased Ctm insertion, both in vitro and in mouse brain, and a strong correlation is found between CtmPrP insertion and neuropathology in transgenic mice; a copper-dependent pathogenicity mechanism is suggested. PrP fusions with a C-terminal epitope tag, when expressed in yeast cells at moderate levels, appear to interact efficiently with the translocon, providing a useful model for testing the effects of PrP mutations on TM insertion and orientation. However, secretion of PrP by the mammalian translocon requires the TRAP complex, absent in yeast, where essentially all PrP ends up as TM species, 85–90% Ntm and 10–15% Ctm. Although yeast is, therefore, an incomplete mimic of mammalian PrP trafficking, effects on Ctm insertion of mutations increasing TM1 hydrophobicity closely reflect those seen in vitro. Electrostatic substrate-translocon interactions are a major determinant of TM protein insertion orientation and the yeast model was used to investigate the role of the large negative charge difference across TM1, a likely cause of translocation delay that would favor TM insertion and Ctm orientation. An increase in ΔCh from −5 to −7 caused a marked increase in Ctm insertion, while a decrease to −3 or −1 allowed 35 and about 65% secretion, respectively. Utility of the yeast model and the role of this charge difference in driving PrP membrane insertion are confirmed.     

Combinatorial control of prion protein biogenesis by the signal sequence and transmembrane domain

The prion protein (PrP) is synthesized in three topologic forms at the endoplasmic reticulum. (sec)PrP is fully translocated into the endoplasmic reticulum lumen, whereas (Ntm)PrP and (Ctm)PrP are single-spanning membrane proteins of opposite orientation. Increased generation of (Ctm)PrP in either transgenic mice or humans is associated with the development of neurodegenerative disease. To study the mechanisms by which PrP can achieve three topologic outcomes, we analyzed the translocation of proteins containing mutations introduced into either the N-terminal signal sequence or potential transmembrane domain (TMD) of PrP. Although mutations in either domain were found to affect PrP topogenesis, they did so in qualitatively different ways. In addition to its traditional role in mediating protein targeting, the signal was found to play a surprising role in determining orientation of the PrP N terminus. By contrast, the TMD was found to influence membrane integration. Analysis of various signal and TMD double mutants demonstrated that the topologic consequence of TMD action was directly dependent on the previous, signal-mediated step. Together, these results reveal that PrP topogenesis is controlled at two discrete steps during its translocation and provide a framework for understanding how these steps act coordinately to determine the final topology achieved by PrP.     

Interactions between the conserved hydrophobic region of the prion protein and dodecylphosphocholine micelles

The three-dimensional structure of PrP110-136, a peptide encompassing the conserved hydrophobic region of the human prion protein, has been determined at high resolution in dodecylphosphocholine micelles by NMR. The results support the conclusion that the (Ctm)PrP, a transmembrane form of the prion protein, adopts a different conformation than the reported structures of the normal prion protein determined in solution. Paramagnetic relaxation enhancement studies with gadolinium-diethylenetriaminepentaacetic acid indicated that the conserved hydrophobic region peptide is not inserted symmetrically in the micelle, thus suggesting the presence of a guanidium-phosphate ion pair involving the side chain of the terminal arginine and the detergent headgroup. Titration of dodecylphosphocholine into a solution of PrP110-136 revealed the presence of a surface-bound species. In addition, paramagnetic probes located the surface-bound peptide somewhere below the micelle-water interface when using the inserted helix as a positional reference. This localization of the unknown population would allow a similar ion pair interaction.     

Membrane topology influences N-glycosylation of the prion protein

The glycosylation state of the glycosyl-phosphatidylinositol (GPI) anchored cellular prion protein (PrPC) can influence the formation of the disease form of the protein responsible for the neurodegenerative spongiform encephalopathies. We have investigated the role of membrane topology in the N-glycosylation of PrP by expressing a C-terminal transmembrane anchored form, PrP-CTM, an N-terminal transmembrane anchored form, PrP-NTM, a double-anchored form, PrP-DA, and a truncated form, PrPDeltaGPI, in human neuroblastoma SH-SY5Y cells. Wild-type PrP, PrP- CTM and PrP-DA were membrane anchored and present on the cell surface as glycosylated forms. In contrast, PrP-NTM, although membrane anchored and localized at the cell surface, was not N-glycosylated. PrPDeltaGPI was secreted from the cells into the medium in a hydrophilic form that was unglycosylated. The 4-fold slower rate at which PrPDeltaGPI was trafficked through the cell compared with wild-type PrP was due to the absence of the GPI anchor not the lack of N-glycans. Retention of PrPDeltaGPI in the endoplasmic reticulum did not lead to its glycosylation. These results indicate that C-terminal membrane anchorage is required for N-glycosylation of PrP.     

Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C terminus

Prion protein (PrP) is synthesized at the membrane of the endoplasmic reticulum (ER) in three different topological forms as follows: a fully translocated one ((sec)PrP) and two with opposite orientations in the membrane ((Ntm)PrP and (Ctm)PrP). We asked whether other signal sequences exist in the PrP, other than the N-terminal signal sequence, that contribute to its topological diversity. In vitro translocation assays showed that PrP lacking its N-terminal signal sequence could still translocate into ER microsomes, although at reduced efficiency. Deletion of each of the two hydrophobic regions in PrP revealed that the C-terminally located hydrophobic region (TM2) can function as second signal sequence in PrP. Translocation mediated by the TM2 alone can occur post-translationally and yields mainly (Ctm)PrP, which is implicated in some forms of neurodegeneration in prion diseases. We conclude that, in vitro, PrP can insert into ER membranes co- and post-translationally and can use two different signal sequences. We propose that the unusually complex topology of PrP results from the differential utilization of two signal sequences in PrP.     

Prion peptide 106-126 modulates the aggregation of cellular prion protein and induces the synthesis of potentially neurotoxic transmembrane PrP.

In infectious and familial prion disorders, neurodegeneration is often seen without obvious deposits of the scrapie prion protein (PrP(Sc)), the principal cause of neuronal death in prion disorders. In such cases, neurotoxicity must be mediated by alternative pathways of cell death. One such pathway is through a transmembrane form of PrP. We have investigated the relationship between intracellular accumulation of prion protein aggregates and the consequent up-regulation of transmembrane prion protein in a cell model. Here, we report that exposure of neuroblastoma cells to the prion peptide 106-126 catalyzes the aggregation of cellular prion protein to a weakly proteinase K-resistant form and induces the synthesis of transmembrane prion protein, the proposed mediator of neurotoxicity in certain prion disorders. The N terminus of newly synthesized transmembrane prion protein is cleaved spontaneously on the cytosolic face of the endoplasmic reticulum, and the truncated C-terminal fragment accumulates on the cell surface. Our results suggest that neurotoxicity in prion disorders is mediated by a complex pathway involving transmembrane prion protein and not by deposits of aggregated and proteinase K-resistant PrP alone.     

Characterization of the properties and trafficking of an anchorless form of the prion protein

Conversion of PrP(C) into PrP(Sc) is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrP Delta GPI). We found that PrP Delta GPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrP(C), this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrP Delta GPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrP(C) and/or of the converted scrapie form might be necessary for the development of a symptomatic disease.     

The low-density lipoprotein receptor-related protein 1 (LRP1) mediates the endocytosis of the cellular prion protein

PrP(C) (cellular prion protein) is located at the surface of neuronal cells in detergent-insoluble lipid rafts, yet is internalized by clathrin-dependent endocytosis. As PrP(C) is glycosyl-phosphatidylinositol-anchored, it requires a transmembrane adaptor protein to connect it to the clathrin endocytosis machinery. Using receptor-associated protein and small interfering RNA against particular LDL (low-density lipoprotein) family members, in combination with immunofluorescence microscopy and surface biotinylation assays, we show that the transmembrane LRP1 (LDL receptor-related protein 1) is required for the Cu(2+)-mediated endocytosis of PrP(C) in neuronal cells. We show also that another LRP1 ligand that can cause neurodegenerative disease, the Alzheimer's amyloid precursor protein, does not modulate the endocytosis of PrP(C).     

Evolving views in prion glycosylation: functional and pathological implications

Prion diseases form a group of neurodegenerative disorders with the unique feature of being transmissible. These diseases involve a pathogenic protein, called PrP(Sc) for the scrapie isoform of the cellular prion protein (PrP(C)) which is an abnormally-folded counterpart of PrP(C). Many questions remain unresolved concerning the function of PrP(C) and the mechanisms underlying prion replication, transmission and neurodegeneration. PrP(C) is a glycosyl-phosphatidylinositol-anchored glycoprotein expressed at the cell surface of neurons and other cell types. PrP(C) may be present as distinct isoforms depending on proteolytic processing (full length and truncated), topology(GPI-anchored, transmembrane or soluble) and glycosylation (non- mono- and di-glycosylated). The present review focuses on the implications of PrP(C) glycosylation as to the function of the normal protein, the cellular pathways of conversion into PrP(Sc), the diversity of prion strains and the related selective neuronal targeting.     

The prion protein and lipid rafts

Prions are the causative agent of the transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrP(C)) undergoes a post-translational conformational conversion to the infectious form (PrP(Sc)). PrP(C) associates with cholesterol- and glycosphingolipid-rich lipid rafts through association of its glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and through interaction of its N-terminal region with an as yet unidentified raft associated molecule. PrP(C) resides in detergent-resistant domains that have different lipid and protein compositions to the domains occupied by another GPI-anchored protein, Thy-1. In some cells PrP(C) may endocytose through caveolae, but in neuronal cells, upon copper binding to the N-terminal octapeptide repeats, the protein translocates out of rafts into detergent-soluble regions of the plasma membrane prior to endocytosis through clathrin-coated pits. The current data suggest that the polybasic region at its N-terminus is required to engage PrP(C) with a transmembrane adaptor protein which in turn links with the clathrin endocytic machinery. PrP(C) associates in rafts with a variety of signalling molecules, including caveolin-1 and Fyn and Src tyrosine kinases. The clustering of PrP(C) triggers a range of signal transduction processes, including the recruitment of the neural cell adhesion molecule to rafts which in turn promotes neurite outgrowth. Lipid rafts appear to be involved in the conformational conversion of PrP(C) to PrP(Sc), possibly by providing a favourable environment for this process to occur and enabling disease progression.     

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP(Sc) produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP(Sc) recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a beta-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP(Sc), and the other to classical PrP(Sc). Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP(Sc) with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.     

Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.