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A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane.

Haigh NG, Johnson AE - J. Cell Biol. (2002)

Bottom Line: Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain.By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration.Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.

ABSTRACT
In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome-translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome-translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.

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Mechanism for maintaining the permeability barrier of the ER membrane during cotranslational membrane protein integration. (a) Prior to integration, the ribosome-free translocon is sealed on the lumenal side by BiP. (b) After SRP-dependent targeting of a ribosome-nascent chain complex to the translocon and translation to yield a nascent chain longer than 70 amino acids (Crowley et al., 1994), the ribosome–translocon seal is intact and the lumenal end of the pore is open (e.g., 111p-86). (c) After the TM sequence has been synthesized and is still near the peptidyltransferase center far inside the ribosome (Liao et al., 1997), the lumenal end of the translocon pore is closed by the action of BiP (111p-88, 111p-91). Although BiP is shown here physically plugging the pore, BiP may effect closure indirectly by binding to another protein(s) that physically closes the pore. At this point, the ribosome–translocon seal is still intact. (d) The ribosome–translocon seal is then broken, whereas the BiP-dependent seal at the other end of the pore remains intact (111p-93). Although the ribosome is depicted here as rotating relative to the translocon, the nature and magnitude of this structural change is not yet known. (e) After termination of translation, the TM sequence is integrated into the ER membrane.
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fig4: Mechanism for maintaining the permeability barrier of the ER membrane during cotranslational membrane protein integration. (a) Prior to integration, the ribosome-free translocon is sealed on the lumenal side by BiP. (b) After SRP-dependent targeting of a ribosome-nascent chain complex to the translocon and translation to yield a nascent chain longer than 70 amino acids (Crowley et al., 1994), the ribosome–translocon seal is intact and the lumenal end of the pore is open (e.g., 111p-86). (c) After the TM sequence has been synthesized and is still near the peptidyltransferase center far inside the ribosome (Liao et al., 1997), the lumenal end of the translocon pore is closed by the action of BiP (111p-88, 111p-91). Although BiP is shown here physically plugging the pore, BiP may effect closure indirectly by binding to another protein(s) that physically closes the pore. At this point, the ribosome–translocon seal is still intact. (d) The ribosome–translocon seal is then broken, whereas the BiP-dependent seal at the other end of the pore remains intact (111p-93). Although the ribosome is depicted here as rotating relative to the translocon, the nature and magnitude of this structural change is not yet known. (e) After termination of translation, the TM sequence is integrated into the ER membrane.

Mentions: However, the ribosomes and translocons in the EM studies clearly do interact in a meaningful way because the ribosomal exit site is localized close to the Sec61 complex in all of the EM images. In fact, the ribosome and the Sec61 complex are aligned by four specific connections that orient them, but leave a gap between them (Beckmann et al., 2001). These interactions are both strong and membrane independent because they survive EM preparation procedures (Beckmann et al., 1997, 2001; Ménétret et al., 2000). Indeed, these same interactions may be responsible for keeping the ribosome near the translocon when the ribosomal seal is broken to allow nascent chain movement into the cytosol during integration (Fig. 4 d). However, because no gap larger than 9 Å (the diameter of a hydrated iodide ion) between the cytoplasm and the ribosomal nascent chain tunnel is detected in the fluorescence experiments with intact membranes and translocons, we conclude that the gap observed in reconstructed EM images of translocation intermediates does not accurately depict all of the ribosome interactions with a complete translocon in an intact membrane.


A new role for BiP: closing the aqueous translocon pore during protein integration into the ER membrane.

Haigh NG, Johnson AE - J. Cell Biol. (2002)

Mechanism for maintaining the permeability barrier of the ER membrane during cotranslational membrane protein integration. (a) Prior to integration, the ribosome-free translocon is sealed on the lumenal side by BiP. (b) After SRP-dependent targeting of a ribosome-nascent chain complex to the translocon and translation to yield a nascent chain longer than 70 amino acids (Crowley et al., 1994), the ribosome–translocon seal is intact and the lumenal end of the pore is open (e.g., 111p-86). (c) After the TM sequence has been synthesized and is still near the peptidyltransferase center far inside the ribosome (Liao et al., 1997), the lumenal end of the translocon pore is closed by the action of BiP (111p-88, 111p-91). Although BiP is shown here physically plugging the pore, BiP may effect closure indirectly by binding to another protein(s) that physically closes the pore. At this point, the ribosome–translocon seal is still intact. (d) The ribosome–translocon seal is then broken, whereas the BiP-dependent seal at the other end of the pore remains intact (111p-93). Although the ribosome is depicted here as rotating relative to the translocon, the nature and magnitude of this structural change is not yet known. (e) After termination of translation, the TM sequence is integrated into the ER membrane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199230&req=5

fig4: Mechanism for maintaining the permeability barrier of the ER membrane during cotranslational membrane protein integration. (a) Prior to integration, the ribosome-free translocon is sealed on the lumenal side by BiP. (b) After SRP-dependent targeting of a ribosome-nascent chain complex to the translocon and translation to yield a nascent chain longer than 70 amino acids (Crowley et al., 1994), the ribosome–translocon seal is intact and the lumenal end of the pore is open (e.g., 111p-86). (c) After the TM sequence has been synthesized and is still near the peptidyltransferase center far inside the ribosome (Liao et al., 1997), the lumenal end of the translocon pore is closed by the action of BiP (111p-88, 111p-91). Although BiP is shown here physically plugging the pore, BiP may effect closure indirectly by binding to another protein(s) that physically closes the pore. At this point, the ribosome–translocon seal is still intact. (d) The ribosome–translocon seal is then broken, whereas the BiP-dependent seal at the other end of the pore remains intact (111p-93). Although the ribosome is depicted here as rotating relative to the translocon, the nature and magnitude of this structural change is not yet known. (e) After termination of translation, the TM sequence is integrated into the ER membrane.
Mentions: However, the ribosomes and translocons in the EM studies clearly do interact in a meaningful way because the ribosomal exit site is localized close to the Sec61 complex in all of the EM images. In fact, the ribosome and the Sec61 complex are aligned by four specific connections that orient them, but leave a gap between them (Beckmann et al., 2001). These interactions are both strong and membrane independent because they survive EM preparation procedures (Beckmann et al., 1997, 2001; Ménétret et al., 2000). Indeed, these same interactions may be responsible for keeping the ribosome near the translocon when the ribosomal seal is broken to allow nascent chain movement into the cytosol during integration (Fig. 4 d). However, because no gap larger than 9 Å (the diameter of a hydrated iodide ion) between the cytoplasm and the ribosomal nascent chain tunnel is detected in the fluorescence experiments with intact membranes and translocons, we conclude that the gap observed in reconstructed EM images of translocation intermediates does not accurately depict all of the ribosome interactions with a complete translocon in an intact membrane.

Bottom Line: Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain.By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration.Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.

ABSTRACT
In mammalian cells, most membrane proteins are inserted cotranslationally into the ER membrane at sites termed translocons. Although each translocon forms an aqueous pore, the permeability barrier of the membrane is maintained during integration, even when the otherwise tight ribosome-translocon seal is opened to allow the cytoplasmic domain of a nascent protein to enter the cytosol. To identify the mechanism by which membrane integrity is preserved, nascent chain exposure to each side of the membrane was determined at different stages of integration by collisional quenching of a fluorescent probe in the nascent chain. Comparing integration intermediates prepared with intact, empty, or BiP-loaded microsomes revealed that the lumenal end of the translocon pore is closed by BiP in an ATP-dependent process before the opening of the cytoplasmic ribosome-translocon seal during integration. This BiP function is distinct from its previously identified role in closing ribosome-free, empty translocons because of the presence of the ribosome at the translocon and the nascent membrane protein that extends through the translocon pore and into the lumen during integration. Therefore, BiP is a key component in a sophisticated mechanism that selectively closes the lumenal end of some, but not all, translocons occupied by a nascent chain. By using collisional quenchers of different sizes, the large internal diameter of the ribosome-bound aqueous translocon pore was found to contract when BiP was required to seal the pore during integration. Therefore, closure of the pore involves substantial conformational changes in the translocon that are coupled to a complex sequence of structural rearrangements on both sides of the ER membrane involving the ribosome and BiP.

Show MeSH
Related in: MedlinePlus