The signal peptide plus a cluster of positive charges in prion protein dictate chaperone-mediated Sec61 channel gating

Anke Ziska; Jörg Tatzelt; Johanna Dudek; Adrienne W. Paton; James C. Paton; Richard Zimmermann; Sarah Haßdenteufel

doi:10.1242/bio.040691

Chaperone-Mediated Sec61 Channel Gating during ER Import of Small Precursor Proteins Overcomes Sec61 Inhibitor-Reinforced Energy Barrier

Cell Reports ◽

10.1016/j.celrep.2018.03.122 ◽

2018 ◽

Vol 23 (5) ◽

pp. 1373-1386 ◽

Cited By ~ 21

Author(s):

Sarah Haßdenteufel ◽

Nicholas Johnson ◽

Adrienne W. Paton ◽

James C. Paton ◽

Stephen High ◽

...

Keyword(s):

Energy Barrier ◽

Channel Gating ◽

Precursor Proteins ◽

Sec61 Channel

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Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

10.1101/867762 ◽

2019 ◽

Author(s):

Stefan Schorr ◽

Duy Nguyen ◽

Sarah Haßdenteufel ◽

Nagarjuna Nagaraj ◽

Adolfo Cavalié ◽

...

Keyword(s):

Signal Peptide ◽

Mammalian Cells ◽

Protein Import ◽

Supplementary Information ◽

Signal Peptides ◽

Proteomics Data ◽

Amino Terminal ◽

Link Type ◽

Sec61 Channel

AbstractIn mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61α or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61α. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease.DatabasesThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).

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A Transmembrane Form of the Prion Protein Contains an Uncleaved Signal Peptide and Is Retained in the Endoplasmic Reticululm

Molecular Biology of the Cell ◽

10.1091/mbc.12.4.881 ◽

2001 ◽

Vol 12 (4) ◽

pp. 881-889 ◽

Cited By ~ 96

Author(s):

Richard S. Stewart ◽

Bettina Drisaldi ◽

David A. Harris

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Prion Protein ◽

Signal Peptide ◽

Mutant Form ◽

Considerable Evidence ◽

Cellular Pathways ◽

Glycolipid Anchor ◽

Infectious Form ◽

Insight Into

Although there is considerable evidence that PrPScis the infectious form of the prion protein, it has recently been proposed that a transmembrane variant calledCtmPrP is the direct cause of prion-associated neurodegeneration. We report here, using a mutant form of PrP that is synthesized exclusively with theCtmPrP topology, thatCtmPrP is retained in the endoplasmic reticulum and is degraded by the proteasome. We also demonstrate thatCtmPrP 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 whichCtmPrP may cause disease.

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A Transmembrane Form of the Prion Protein Is Localized in the Golgi Apparatus of Neurons

Journal of Biological Chemistry ◽

10.1074/jbc.m412298200 ◽

2005 ◽

Vol 280 (16) ◽

pp. 15855-15864 ◽

Cited By ~ 33

Author(s):

Richard S. Stewart ◽

David A. Harris

Keyword(s):

Golgi Apparatus ◽

Prion Protein ◽

Cell Lines ◽

Signal Peptide ◽

Transmembrane Domain ◽

Cerebellar Granule Neurons ◽

Granule Neurons ◽

Cerebellar Granule ◽

Neurotoxic Effects ◽

Neuronal Processes

CtmPrP 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 theCtmPrP form in transfected cell lines. To characterize the properties ofCtmPrP 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 representsCtmPrP, with the rest beingSecPrP, the glycolipid anchored form that does not span the membrane. Both forms contained an uncleaved signal peptide, and they are differentially glycosylated.SecPrP was localized on the surface of neuronal processes. Most surprisingly,CtmPrP 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 ofCtmPrP in a neuronal context, and our results suggest new hypotheses about how this form may exert its neurotoxic effects.

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Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane

Journal of Cell Science ◽

10.1242/jcs.112.23.4185 ◽

1999 ◽

Vol 112 (23) ◽

pp. 4185-4191 ◽

Cited By ~ 5

Author(s):

K. Romisch

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Proteins ◽

Signal Peptide ◽

Protein Translocation ◽

Transmembrane Proteins ◽

Retrograde Transport ◽

Conducting Channel ◽

Peptide Cleavage ◽

Sec61 Channel ◽

Er Membrane

Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded. Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol. The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane. For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane. Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.

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Metallic prions

Biochemical Society Symposium ◽

10.1042/bss0710193 ◽

2004 ◽

Vol 71 ◽

pp. 193-202 ◽

Cited By ~ 9

Author(s):

David R Brown

Keyword(s):

Prion Protein ◽

Binding Capacity ◽

Normal Function ◽

Transmissible Spongiform Encephalopathies ◽

Published Data ◽

Normal Brain ◽

Copper Binding ◽

Loss Of Function ◽

Spongiform Encephalopathies ◽

The Brain

Prion diseases, also referred to as transmissible spongiform encephalopathies, are characterized by the deposition of an abnormal isoform of the prion protein in the brain. However, this aggregated, fibrillar, amyloid protein, termed PrPSc, is an altered conformer of a normal brain glycoprotein, PrPc. Understanding the nature of the normal cellular isoform of the prion protein is considered essential to understanding the conversion process that generates PrPSc. To this end much work has focused on elucidation of the normal function and activity of PrPc. Substantial evidence supports the notion that PrPc is a copper-binding protein. In conversion to the abnormal isoform, this Cu-binding activity is lost. Instead, there are some suggestions that the protein might bind other metals such as Mn or Zn. PrPc functions currently under investigation include the possibility that the protein is involved in signal transduction, cell adhesion, Cu transport and resistance to oxidative stress. Of these possibilities, only a role in Cu transport and its action as an antioxidant take into consideration PrPc's Cu-binding capacity. There are also more published data supporting these two functions. There is strong evidence that during the course of prion disease, there is a loss of function of the prion protein. This manifests as a change in metal balance in the brain and other organs and substantial oxidative damage throughout the brain. Thus prions and metals have become tightly linked in the quest to understand the nature of transmissible spongiform encephalopathies.

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