Cotranslational Membrane Protein Biogenesis at the Endoplasmic Reticulum

Alder, Nathan N.; Johnson, Arthur E.

doi:10.1074/jbc.r400002200

Cited by 119 publications

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“…A number of studies by different groups have demonstrated that a signal sequence or a signal-anchor (SA) sequence (an uncleaved signal sequence that is sufficiently nonpolar to integrate and form a TMS in the bilayer) is transiently adjacent to translocon proteins after SRP-dependent targeting (1,3). To determine whether the SM sequence of an INM protein also passes through the translocon, parallel samples of E66 mRNAs containing amber codons at positions 10, 11, 12, or 13 were translated in the presence of SRP, microsomes, and ANB-Lys-tRNA amb , resulting in a 70-residue nascent chain (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…M ost membrane proteins in eukaryotic cells are cotranslationally integrated into the membrane of the endoplasmic reticulum (ER) at sites termed translocons (1)(2)(3). These proteins are then sorted and distributed to cellular locations where they function, typically, by means of vesicular trafficking to the Golgi compartments, other organelles, and the plasma membrane (4,5).…”

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confidence: 99%

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Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane

Saksena

Shao

Braunagel

et al. 2004

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

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M ost membrane proteins in eukaryotic cells are cotranslationally integrated into the membrane of the endoplasmic reticulum (ER) at sites termed translocons (1-3). These proteins are then sorted and distributed to cellular locations where they function, typically, by means of vesicular trafficking to the Golgi compartments, other organelles, and the plasma membrane (4, 5). However, in some cases, newly synthesized membrane proteins are directed to the inner nuclear membrane (INM). Because the INM is contiguous with the ER membrane, it is generally presumed that, after leaving the translocon, membrane proteins destined for the INM diffuse through the ER membrane, the outer nuclear membrane, and the nuclear pore membrane to reach the INM (6). Proteins are then retained in the INM by binding to nuclear proteins or DNA that prevent their diffusion back into the ER membrane. This model for protein sorting to the INM is termed the ''diffusion-retention'' model (6).The envelope proteins of the baculovirus occlusion-derived virus (ODV) also integrate into the ER and transit to virally induced intranuclear membranes for envelope assembly. A minimum sequence required to direct proteins to the INM was determined by using the viral envelope protein ODV-E66 (E66), and its N-terminal 33 aa were found to be sufficient to traffic fusion proteins to the INM and ODV envelope with an efficiency similar to wild-type protein (7). This sequence has therefore been termed an INM sorting motif (SM) (8). The SM consists of 18 hydrophobic amino acids that form a transmembrane sequence (TMS) with positively charged amino acids that are located four to eight residues from the TMS on the nucleoplasmic or cytoplasmic face of the membrane. Notably, the SM also directs proteins to the INM in the absence of infection (8). Furthermore, a comparison of the viral SM sequence with the sequences of cellular proteins revealed that well-characterized cellular INM proteins have an SM-like sequence, even though the TMSs may be oriented in opposite directions in the bilayer and may be inserted into the bilayer by different mechanisms (8). Hence, fusion proteins containing either viral or cellular SM sequences may constitute reasonable substrates for examining the molecular mechanisms that mediate protein movement into the INM.Although diffusion-retention could explain ODV envelope protein trafficking, transport to the INM may be regulated by something more complex than passive diffusion and retention. The 33-residue E66 SM sequence was crosslinked to FP25K and BV͞ODV-E26 (E26), thereby demonstrating that these two viral proteins are adjacent to the SM while it is still in the ER membrane (8). Moreover, when the gene coding for FP25K was deleted from the viral genome, trafficking of E66 during infection seemed to be blocked at the nuclear envelope: E66 accumulated in punctate regions associated with the ONM and was not detected in the INM or within the virally induced intranuclear membranes (9). Thus, the trafficking of E66 to the INM may involve the activ...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane

Saksena

Shao

Braunagel

et al. 2004

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

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“…The polypeptide chain of the secretory protein is translocated through the pore (5). On the other hand, a large aqueous environment of the translocon has been observed (6). Moreover, the translocon possesses such remarkable flexibility that two translocating hydrophilic polypeptide chains can be simultaneously accommodated (7).…”

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Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

Kida

Morimoto

Sakaguchi

2009

Journal of Biological Chemistry

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Many proteins are translocated across and integrated into the endoplasmic reticulum membrane. The type I signal anchor sequence mediates the translocation of its preceding region through the endoplasmic reticulum membrane, but the source of the motive force has been unclear. Here, we characterized the motive force for N-terminal domain translocation using two probes. First, an Ig-like domain of the muscle protein titin (I27 domain) or its mutants were fused to the N termini, and translocation was examined in a cell-free translation system supplemented with rough microsomal membrane. The N-terminal translocation efficiencies correlated with the mechanical instabilities of the I27 mutants. When the I27 domain was separated from the signal anchor sequence by inserting a spacer, even the most unstable mutant stalled on the cytoplasmic side, whereas its downstream portion spanned the membrane. Proline insertion into the signal anchor sequence also caused a large translocation defect. Second, a streptavidin-binding peptide tag was fused to the N terminus. Titration of streptavidin in the translation system allowed us to estimate the translocation motive force operative on the tag. The motive force was decreased by the proline insertion into the signal anchor sequence as well as by separation from the signal anchor sequence. When the streptavidin-binding peptide tag was separated from the signal anchor, the proline insertion did not induce further deficits in the motive force for the tag. On the basis of the findings obtained by using these two independent techniques, we conclude that the signal sequence itself provides the motive force for N-terminal domain translocation within a limited upstream region.

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“…Membrane protein assembly in both eukaryotes and prokaryotes is initiated by the cotranslational targeting of ribosome-associated nascent chains (RNCs) 3 to the Sec translocons in the endoplasmic reticulum or the bacterial cytoplasmic membrane. This requires binding of the signal recognition particle (SRP) to the signal anchor sequence of a membrane protein when it emerges from the ribosomal exit tunnel and the subsequent interaction of the SRP-RNC complex with the membrane-attached SRP receptor (SR) (1,2).…”

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“…This requires binding of the signal recognition particle (SRP) to the signal anchor sequence of a membrane protein when it emerges from the ribosomal exit tunnel and the subsequent interaction of the SRP-RNC complex with the membrane-attached SRP receptor (SR) (1,2). The subsequent transfer of the RNC to the Sec translocon is probably favored by the proposed close vicinity of the SR to the Sec translocon (3). A direct interaction between FtsY, the bacterial SR, and SecY has recently been demonstrated in Escherichia coli (4).…”

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A Dual Function for SecA in the Assembly of Single Spanning Membrane Proteins in Escherichia coli

Deitermann

Sprie

Koch

2005

Journal of Biological Chemistry

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The assembly of bacterial membrane proteins with large periplasmic loops is an intrinsically complex process because the SecY translocon has to coordinate the signal recognition particledependent targeting and integration of transmembrane domains with the SecA-dependent translocation of the periplasmic loop. The current model suggests that the ATP hydrolysis by SecA is required only if periplasmic loops larger than 30 amino acids have to be translocated. In agreement with this model, our data demonstrate that the signal recognition particle-and SecA-dependent multiple spanning membrane protein YidC becomes SecA-independent if the large periplasmic loop connecting transmembrane domains 1 and 2 is reduced to less than 30 amino acids. Strikingly, however, we were unable to render single spanning membrane proteins SecAindependent by reducing the length of their periplasmic loops. For these proteins, the complete assembly was always SecA-dependent even if the periplasmic loop was reduced to 13 amino acids. If, however, the 13-amino acid-long periplasmic loop was fused to a downstream transmembrane domain, SecA was no longer required for complete translocation. Although these data support the current model on the SecA dependence of multiple spanning membrane proteins, they indicate a novel function of SecA for the assembly of single spanning membrane proteins. This could suggest that single and multiple spanning membrane proteins are processed differently by the bacterial SecY translocon.Membrane protein assembly in both eukaryotes and prokaryotes is initiated by the cotranslational targeting of ribosome-associated nascent chains (RNCs) 3 to the Sec translocons in the endoplasmic reticulum or the bacterial cytoplasmic membrane. This requires binding of the signal recognition particle (SRP) to the signal anchor sequence of a membrane protein when it emerges from the ribosomal exit tunnel and the subsequent interaction of the SRP-RNC complex with the membrane-attached SRP receptor (SR) (1, 2). The subsequent transfer of the RNC to the Sec translocon is probably favored by the proposed close vicinity of the SR to the Sec translocon (3). A direct interaction between FtsY, the bacterial SR, and SecY has recently been demonstrated in Escherichia coli (4). In contrast to the eukaryotic SRP, which targets both membrane and secretory proteins, the bacterial SRP is predominantly engaged in membrane protein targeting. The vast majority of bacterial secretory proteins, i.e. proteins that are destined to reach the periplasmic space or the outer membrane, are post-translationally targeted by the bacteria-specific SecA/SecB pathway (5). In this pathway, SecB functions as a secretion-specific chaperone for most secretory proteins, whereas SecA is proposed to translocate the preprotein in a stepwise translocation of stretches of about 30 amino acids (6 -8).The translocation of large luminal domains in eukaryotic membrane proteins does not depend on cytosolic proteins other than SRP (3). This is different for bacterial membrane prote...

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Cotranslational Membrane Protein Biogenesis at the Endoplasmic Reticulum

Cited by 119 publications

References 50 publications

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane

Signal Anchor Sequence Provides Motive Force for Polypeptide Chain Translocation through the Endoplasmic Reticulum Membrane

A Dual Function for SecA in the Assembly of Single Spanning Membrane Proteins in Escherichia coli

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