Reduced translocation of nascent prion protein during ER stress contributes to neurodegeneration

During acute stress in the endoplasmic reticulum (ER), mammalian prion protein (PrP) is temporarily prevented from translocation into the ER and instead routed directly for cytosolic degradation. This "pre-emptive" quality control (pQC) system benefits cells by minimizing PrP aggregation in the secretory pathway during ER stress. However, the potential toxicity of cytosolic PrP raised the possibility that persistent pQC of PrP contributes to neurodegeneration in prion diseases. Here, we find evidence of ER stress and decreased translocation of nascent PrP during prion infection. Transgenic mice expressing a PrP variant with reduced translocation at levels expected during ER stress was sufficient to cause several mild age-dependent clinical and histological manifestations of PrP-mediated neurodegeneration. Thus, an ordinarily adaptive quality-control pathway can be contextually detrimental over long time periods. We propose that one mechanism of prion-mediated neurodegeneration involves an indirect ER stress-dependent effect on nascent PrP biosynthesis and metabolism.     

A model for co-translational translocation: ribosome-regulated nascent polypeptide translocation at the protein-conducting channel

The protein-conducting channel (PCC) must allow both the translocation of soluble polypeptide regions across, and the lateral partitioning of hydrophobic transmembrane helices (TMHs) into, the membrane. We have analyzed existing structures of ribosomes and ribosome-PCC complexes and observe conformational changes suggesting that the ribosome may sense and orient the nascent polypeptide and also facilitate conformational changes in the PCC, subsequently directing the nascent polypeptide into the appropriate PCC-mediated translocation mode. The PCC is predicted to be able to accommodate one central, consolidated channel or two segregated pores with different lipid accessibilities, which may enable the lipid-mediated partitioning of a TMH from one pore, while the other, aqueous, pore allows translocation of a hydrophilic polypeptide segment. Our hypothesis suggests a plausible mechanism for the transitioning of the PCC between different configurations.     

Structural insight into the protein translocation channel

A structurally conserved protein translocation channel is formed by the heterotrimeric Sec61 complex in eukaryotes, and SecY complex in archaea and bacteria. Electron microscopy studies suggest that the channel may function as an oligomeric assembly of Sec61 or SecY complexes. Remarkably, the recently determined X-ray structure of an archaeal SecY complex indicates that the pore is located at the center of a single molecule of the complex. This structure suggests how the pore opens perpendicular to the plane of the membrane to allow the passage of newly synthesized secretory proteins across the membrane and opens laterally to allow transmembrane segments of nascent membrane proteins to enter the lipid bilayer. The electron microscopy and X-ray results together suggest that only one copy of the SecY or Sec61 complex within an oligomer translocates a polypeptide chain at any given time.     

Cotranslational protein integration into the ER membrane is mediated by the binding of nascent chains to translocon proteins

During cotranslational protein integration into the ER membrane, each transmembrane (TM) segment moves laterally through the translocon to reach the lipid bilayer. Photocrosslinking studies reveal that a particular surface of each nascent chain TM alpha helix and signal-anchor sequence always faces Sec61alpha in the translocon. This nonrandom and TM sequence-dependent positioning reveals that each TM segment makes specific contacts with Sec61alpha and is retained at a fixed location within the translocon, observations that are best explained by the binding of each TM sequence to a translocon protein(s). Since TM sequence hydrophobicity does not correlate with its rate of release from the translocon, nascent chain movement through the translocon appears to be mediated primarily by protein-protein interactions rather than hydrophobic nascent chain-phospholipid interactions.     

Specific features of the prion protein transmembrane domain regulate nascent chain orientation

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.     

SecYEG assembles into a tetramer to form the active protein translocation channel

Translocase mediates preprotein translocation across the Escherichia coli inner membrane. It consists of the SecYEG integral membrane protein complex and the peripheral ATPase SecA. Here we show by functional assays, negative-stain electron microscopy and mass measurements with the scanning transmission microscope that SecA recruits SecYEG complexes to form the active translocation channel. The active assembly of SecYEG has a side length of 10.5 nm and exhibits an approximately 5 nm central cavity. The mass and structure of this SecYEG as well as the subunit stoichiometry of SecA and SecY in a soluble translocase-precursor complex reveal that translocase consists of the SecA homodimer and four SecYEG complexes.     

Protection from cytosolic prion protein toxicity by modulation of protein translocation

Failure to promptly dispose of undesirable proteins is associated with numerous diseases. In the case of cellular prion protein (PrP), inhibition of the proteasome pathway can generate a highly aggregation-prone, cytotoxic form of PrP implicated in neurodegeneration. However, the predominant mechanisms that result in delivery of PrP, ordinarily targeted to the secretory pathway, to cytosolic proteasomes have been unclear. By accurately measuring the in vivo fidelity of protein translocation into the endoplasmic reticulum (ER), we reveal a slight inefficiency in PrP signal sequence function that generates proteasomally degraded cytosolic PrP. Attenuating this source of cytosolic PrP completely eliminates the dependence on proteasomes for PrP degradation. This allows cells to tolerate both higher expression levels and decreased proteasomal capacity without succumbing to the adverse consequences of misfolded PrP. Thus, the generation of potentially toxic cytosolic PrP is controlled primarily during its initial translocation into the ER. These results suggest that a substantial proportion of the cell's constitutive proteasomal burden may consist of proteins that, like PrP, fail to cotranslationally enter the secretory pathway with high fidelity.     

Cotranslational attachment of fatty acids to nascent peptides in gastric mucus glycoprotein

Using gastric mucous cells which are involved exclusively in the synthesis of secretory O-glycosidic glycoprotein (mucin), the relationship between protein core synthesis and its acylation with fatty acids was investigated. Labeling of the cells with [3H]palmitic acid and [35S]methionine followed by isolation of peptidyl-tRNA and release of nascent peptides, indicated that these peptides contain covalently bound fatty acids. The high performance thin layer chromatography, SDS-gel electrophoresis, and radioactivity scanning revealed that the preparation contained three fractions labeled with palmitate (Mr 15,000-3,600) and two (Mr 1,500 and less) without this label. Based on these data and the nascent peptides amino acid analysis, we conclude that the protein core of the O-glycosidic glycoprotein is acylated with fatty acids during translation, when the peptide chain is longer than 21 amino acid residues.     

NrCAM coupling to the cytoskeleton depends on multiple protein domains and partitioning into lipid rafts

NrCAM is a cell adhesion molecule of the L1 family that is implicated in the control of axonal growth. Adhesive contacts may promote advance of the growth cone by triggering the coupling of membrane receptors with the F-actin retrograde flow. We sought to understand the mechanisms leading to clutching the F-actin at the site of ligand-mediated clustering of NrCAM. Using optical tweezers and single particle tracking of beads coated with the ligand TAG-1, we analyzed the mobility of NrCAM-deletion mutants transfected in a neuroblastoma cell line. Deletion of the cytoplasmic tail did not prevent the coupling of NrCAM to the actin flow. An additional deletion of the FNIII domains to remove cis-interactions, was necessary to abolish the rearward movement of TAG-1 beads, which instead switched to a stationary behavior. Next, we showed that the actin-dependent retrograde movement of NrCAM required partitioning into lipid rafts as indicated by cholesterol depletion experiments using methyl-beta-cyclodextrin. Recruitment of the raft component caveolin-1 was induced at the adhesive contact between the cell surface and TAG-1 beads, indicating that enlarged rafts were generated. Photobleaching experiments showed that the lateral mobility of NrCAM increased with raft dispersion in these contact areas, further suggesting that TAG-1-coated beads induced the coalescence of lipid rafts. In conclusion, we propose that anchoring of NrCAM with the retrograde actin flow can be triggered by adhesive contacts via cooperative processes including interactions with the cytoplasmic tail, formation of cis-complex via the FNIII repeats, and lipid raft aggregation.     

Concurrent translocation of multiple polypeptide chains through the proteasomal degradation channel

The proteasome can actively unfold proteins by sequentially unraveling their substrates from the attachment point of the degradation signal. To investigate the steric constraints imposed on substrate proteins during their degradation by the proteasome, we constructed a model protein in which specific parts of the polypeptide chain were covalently connected through disulfide bridges. The cross-linked model proteins were fully degraded by the proteasome, but two or more cross-links retarded the degradation slightly. These results suggest that the pore of the proteasome allows the concurrent passage of at least three stretches of a polypeptide chain. A degradation channel that can tolerate some steric bulk may reconcile the two opposing needs for degradation that is compartmentalized to avoid aberrant proteolysis yet able to handle a range of substrates of various sizes.     

Mechanisms of protein translocation into mitochondria

Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins. At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified. These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix. The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components. Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP. The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase. The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex. A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins.     

Energetics of protein translocation into mitochondria

Biogenesis of mitochondria depends on the coordinated action of at least six protein translocases present in both mitochondrial membranes. They use different energy sources to drive unidirectional transport of proteins across and into mitochondrial membranes. Here we present an overview on the energetic requirements of different mitochondrial import pathways.     

Folding of the nascent peptide chain into a biologically active protein

The refolding of denatured proteins with complete sequences may not be fast enough to account for the in vivo folding of growing peptide chains during biosynthesis. As some peptide fragments have secondary structures not unlike those of the corresponding segments in the intact molecules and native disulfide bonds of some proteins can form cotranslationally, it is suggested that the folding of the nascent chain begins early during synthesis. However, further adjustments may be necessary during chain elongation and after posttranslational modifications of the completed peptide chain to generate the native conformation of a biologically active protein.     

DNA converts cellular prion protein into the beta-sheet conformation and inhibits prion peptide aggregation

The main hypothesis for prion diseases proposes that the cellular protein (PrP(C)) can be altered into a misfolded, beta-sheet-rich isoform (PrP(Sc)), which in most cases undergoes aggregation. In an organism infected with PrP(Sc), PrP(C) is converted into the beta-sheet form, generating more PrP(Sc). We find that sequence-specific DNA binding to recombinant murine prion protein (mPrP-(23-231)) converts it from an alpha-helical conformation (cellular isoform) into a soluble, beta-sheet isoform similar to that found in the fibrillar state. The recombinant murine prion protein and prion domains bind with high affinity to DNA sequences. Several double-stranded DNA sequences in molar excess above 2:1 (pH 4.0) or 0.5:1 (pH 5.0) completely inhibit aggregation of prion peptides, as measured by light scattering, fluorescence, and circular dichroism spectroscopy. However, at a high concentration, fibers (or peptide aggregates) can rescue the peptide bound to the DNA, converting it to the aggregating form. Our results indicate that a macromolecular complex of prion-DNA may act as an intermediate for the formation of the growing fiber. We propose that host nucleic acid may modulate the delicate balance between the cellular and the misfolded conformations by reducing the protein mobility and by making the protein-protein interactions more likely. In our model, the infectious material would act as a seed to rescue the protein bound to nucleic acid. Accordingly, DNA would act on the one hand as a guardian of the Sc conformation, preventing its propagation, but on the other hand may catalyze Sc conversion and aggregation if a threshold level is exceeded.     

Pathway complexity of prion protein assembly into amyloid

In vivo under pathological conditions, the normal cellular form of the prion protein, PrP(C) (residues 23-231), misfolds to the pathogenic isoform PrP(Sc), a beta-rich aggregated pathogenic multimer. Proteinase K digestion of PrP(Sc) leads to a proteolytically resistant core, PrP 27-30 (residues 90-231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP(Sc) and found that it can adopt two non-native abnormal isoforms, a beta-oligomer and an amyloid fibril. Several lines of kinetic data suggest that the beta-oligomer is not on the pathway to amyloid formation. The preferences for forming either a beta-oligomer or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring the beta-oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high beta-sheet content and bind 1-anilinonaphthalene-8-sulfonate, they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct beta-sheet-rich abnormal isoforms may explain the difficulties in refolding PrP(Sc) in vitro, the need for a PrP(Sc) template, and the significant variation in disease presentation and neuropathology.     

Investigating the SecY plug movement at the SecYEG translocation channel

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.     

A complex of chaperones and disulfide isomerases occludes the cytosolic face of the translocation protein Sec61p and affects translocation of the prion protein

Secretion of newly synthesized proteins across the mammalian rough endoplasmic reticulum (translocation) is supported by the membrane proteins Sec61p and TRAM, but may also include accessory factors, depending on the particular translocation substrate. Studies designed to investigate the binding of anti-peptide antibodies to the carboxyl terminus of the alpha-subunit of Sec61 (Sec61palpha) lead us to the isolation of a complex of proteins that occlude the cytosolic face of Sec61palpha in microsomes that have been prepared by standard protocols used to study translocation in vitro [Walter, P., and Blobel, G. (1983) Methods Enzymol. 96, 84-93]. This complex was shown by nanospray tandem mass spectrometry to be composed of protein disulfide isomerase (PDI), calcium binding protein 1 (CABP1/P5), 72 kDa endoplasmic reticulum protein (ERp72), and BiP (heat shock protein A5/HSPA5), and has been named TR-PDI for "translocon-resident protein disulfide isomerase complex". This constitutes a novel location for these proteins, which are known to be major constituents of the lumen of the rough endoplasmic reticulum. We have not established the function of TR-PDI at this location, but did observe that the absence of this complex results in a relative loss of correct topology of prion protein insertion across RER membranes, indicating the possibility of a functional role in vivo.     

Energy dependence of protein translocation into chloroplasts

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP-dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast.