The Oligomeric Distribution of SecYEG is Altered by SecA and Translocation Ligands

Scheuring, Johannes; Braun, Nathalie; Nothdurft, Lars; Stumpf, Matthias; Veenendaal, Andreas K. J.; Kol, Stefan; Does, Chris van der; Driessen, Arnold J. M.; Weinkauf, Sevil

doi:10.1016/j.jmb.2005.09.058

Cited by 53 publications

(53 citation statements)

References 55 publications

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“…Freeze-fracture experiments with proteoliposomes support the idea that the formation of Sec61-oligomers may be induced by ribosome binding (Hanein et al, 1996). Different quaternary states of the bacterial homologue SecYEG in the membrane have also been described, and their relative abundance may change during translocation (Bessonneau et al, 2002;Scheuring et al, 2005).…”

Section: Discussionmentioning

confidence: 79%

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Schaletzky

Rapoport

2006

MBoC

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We have addressed how ribosome-nascent chain complexes (RNCs), associated with the signal recognition particle (SRP), can be targeted to Sec61 translocation channels of the endoplasmic reticulum (ER) membrane when all binding sites are occupied by nontranslating ribosomes. These competing ribosomes are known to be bound with high affinity to tetramers of the Sec61 complex. We found that the membrane binding of RNC-SRP complexes does not require or cause the dissociation of prebound nontranslating ribosomes, a process that is extremely slow. SRP and its receptor target RNCs to a free population of Sec61 complex, which associates with nontranslating ribosomes only weakly and is conformationally different from the population of ribosome-bound Sec61 complex. Taking into account recent structural data, we propose a model in which SRP and its receptor target RNCs to a Sec61 subpopulation of monomeric or dimeric state. This could explain how RNC-SRP complexes can overcome the competition by nontranslating ribosomes. INTRODUCTIONCotranslational translocation is the major pathway in mammals by which proteins are transported across or integrated into the endoplasmic reticulum (ER) membrane. The synthesis of these proteins starts with a free ribosome in the cytosol. Once a hydrophobic signal sequence or transmembrane (TM) segment has emerged from the ribosome, the signal recognition particle (SRP) binds, forming a ribosomenascent chain (RNC)-SRP complex (for reviews, see Walter and Johnson, 1994;Egea et al., 2005). This complex binds to the ER membrane by interactions between SRP and the SRP receptor (SR) and between the ribosome and the Sec61 complex, a heterotrimeric membrane protein that forms a proteinconducting channel (Van den Berg et al., 2004). The nascent polypeptide chain forms a loop when inserting into the channel, with the signal or TM sequence intercalated into the walls of the channel and the following mature region in the pore proper (for reviews, see Rapoport et al., 2004;Osborne et al., 2005). For secretory proteins, this part of the elongating chain is moved through the channel into the ER lumen, and the signal sequence is eventually cleaved off. In membrane proteins, TM sequences are partitioned sideways through the walls of the channel into the lipid phase, leaving some parts of the polypeptide chain in the cytosol and others in the ER lumen. For both classes of proteins, the translating ribosome stays bound to the channel during the entire translocation process (Mothes et al., 1997).Previous work has shown that the Sec61 complex can bind not only RNCs but also nontranslating ribosomes with nanomolar affinity (Borgese et al., 1974;Kalies et al., 1994;Prinz et al., 2000a). Ribosomes remain bound to ER membranes after nascent chain release (Adelman et al., 1973) and do not easily dissociate from the membrane (Borgese et al., 1973;Potter and Nicchitta, 2002). Given that the concentration of free ribosomes in a secretory cell is ϳ0.3 M, 95% of the Sec61 binding sites should be occupied (Blobel and Potter, ...

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Section: Discussionmentioning

confidence: 79%

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Schaletzky

Rapoport

2006

MBoC

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“…This finding has been confirmed by thiol-cross-linking (11,12) and by cryoelectron microscopy analysis of the eukaryotic Sec complex bound to a translating ribosome (13). It is thus well-established that a single SecY copy is sufficient to form the translocation pathway, yet it remains mysterious that the channel exists as oligomers (14)(15)(16)(17).…”

mentioning

confidence: 81%

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

Dalal

Chan

Sligar

et al. 2012

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

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The SecA ATPase associates with the SecY complex to push preproteins across the bacterial membrane. Because a single SecY is sufficient to create the conducting channel, the function of SecY oligomerization remains unclear. Here, we have analyzed the translocation reaction using nanodiscs. We show that one SecY copy is sufficient to bind SecA and the preprotein, but only the SecY dimer together with acidic lipids supports the activation of the SecA translocation ATPase. In discs, the dimer is predominantly arranged in a back-to-back manner and remains active even if a constituent SecY copy is defective for SecA binding. In membrane vesicles and in intact cells, the coproduction of two inactive SecYs, one for channel gating and the other for SecA binding, recreates a functional translocation unit. These results indisputably argue that the SecY dimer is crucial for the activation of SecA, which is necessary for preprotein transport.

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“…Binding of SecA induces oligomerization of SecYEG protomers, 15,16 and once bound to SecYEG, SecA undergoes multiple conformational changes that ultimately result in translocation of the preproteins. During these cycles of conformational changes, SecA is thought to insert (partially) into the oligomeric SecYEG complex.…”

Section: Introductionmentioning

confidence: 99%

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

Sluis¹,

Nouwen²,

Koch³

et al. 2006

Journal of Molecular Biology

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The Oligomeric Distribution of SecYEG is Altered by SecA and Translocation Ligands

Cited by 53 publications

References 55 publications

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

Two copies of the SecY channel and acidic lipids are necessary to activate the SecA translocation ATPase

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

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