The Aqueous Pore through the Translocon Has a Diameter of 40–60 Å during Cotranslational Protein Translocation at the ER Membrane

Hamman, Brian D.; Chen, Jui‐Chang; Johnson, Edward E; Johnson, Arthur E.

doi:10.1016/s0092-8674(00)80235-4

Cited by 267 publications

(201 citation statements)

References 36 publications

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“…Post-encapsulation, quenching studies reveal that the FD10 present within the DNA cage is inaccessible to particles >3 nm in size, completely accessible to particles below 2 nm, whereas particles between 2 and 3 nm have limited access. Such size-dependent quenching has been observed in the case of the fluorophores present inside the endoplasmic reticulum where the transition point corresponds to the average pore size of the translocon, the protein conduction channel present on the endoplasmic reticulum 33 . Given that the pore size of I has been shown to be ~2.5 nm experimentally and 2.8 nm by a theoretical model 22 , these studies confirm that FD10 is encapsulated as cargo within the DNA icosahedron as a host in the I FD10 complex.…”

Section: Discussionmentioning

confidence: 90%

A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging

et al. 2011

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The encapsulation of molecular cargo within well-defined supramolecular architectures is highly challenging. Synthetic hosts are desirable because of their well-defined nature and addressability. Encapsulation of biomacromolecules within synthetic hosts is especially challenging because of the former's large size, sensitive nature, retention of functionality postencapsulation and demonstration of control over the cargo. Here we encapsulate a fluorescent biopolymer that functions as a pH reporter within synthetic, DNA-based icosahedral host without molecular recognition between host and cargo. Only those cells bearing receptors for the DNA casing of the host-cargo complex engulf it. We show that the encapsulated cargo is therefore uptaken cell specifically in Caenorhabditis elegans. Retention of functionality of the encapsulated cargo is quantitatively demonstrated by spatially mapping pH changes associated with endosomal maturation within the coelomocytes of C. elegans. This is the first demonstration of functionality and emergent behaviour of a synthetic host-cargo complex in vivo.

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Section: Discussionmentioning

confidence: 90%

A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging

et al. 2011

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“…The first hypothesis would require a disassembly reaction catalyzed by proteins presently unknown; the second model presupposes a translocation pore, the Sec61 complex, of a diameter sufficient to allow passage of the US2͞HLA-A2 complex (10). Estimates for the diameter of the Sec61 translocon range from 20 Å as determined by electron cryomicroscopy-based methods (46), to 40-60 Å, based on fluorescence quenching and electrophysiological measurements (47). The upper size estimate of the Sec61 pore might allow dislocation of the entire US2͞HLA-A2 (the entire complex could fit in a rectangular box of approximate dimensions 50 ϫ 70 ϫ 80 Å 3 ).…”

Section: Us2 Binding Site Is Remote From Known Class I͞peptide-loadingmentioning

confidence: 99%

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Gewurz

Gaudet

Tortorella

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

139

134

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Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cellsurface expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2͞Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the ␣3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.

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“…On the other hand, experimental studies with microsomes harboring the eukaryotic This article is a PNAS Direct Submission. Sec61 complex indicate a pore diameter of 40-60 Å in the active state (17). The SecYEG complex seems rather promiscuous as it can translocate preproteins that are chemically cross-linked to nonpolypeptide constituents (18,19).…”

mentioning

confidence: 99%

Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules

Bonardi

Halža

Walko

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Protein translocation in Escherichia coli is mediated by the translocase that in its minimal form consists of the protein-conducting channel SecYEG, and the motor protein, SecA. SecYEG forms a narrow pore in the membrane that allows passage of unfolded proteins only. Molecular dynamics simulations suggest that the maximal width of the central pore of SecYEG is limited to 16 Å. To access the functional size of the SecYEG pore, the precursor of outer membrane protein A was modified with rigid spherical tetraarylmethane derivatives of different diameters at a unique cysteine residue. SecYEG allowed the unrestricted passage of the precursor of outer membrane protein A conjugates carrying tetraarylmethanes with diameters up to 18 Å, whereas a 29 Å sized molecule blocked the translocation pore. Translocation of the protein-organic molecule hybrids was strictly proton motive force-dependent and occurred at a single pore. With an average diameter of an unfolded polypeptide chain of 4-6 Å, the pore accommodates structures of at least 22-24 Å, which is vastly larger than the predicted maximal width of a single pore by molecular dynamics simulations.secretion | Sec-system

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The Aqueous Pore through the Translocon Has a Diameter of 40–60 Å during Cotranslational Protein Translocation at the ER Membrane

Cited by 267 publications

References 36 publications

A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging

A synthetic icosahedral DNA-based host–cargo complex for functional in vivo imaging

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

Probing the SecYEG translocation pore size with preproteins conjugated with sizable rigid spherical molecules

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