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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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Iodide ion quenching of NBD-labeled 111p-91 integration intermediates prepared with various ER microsomes. Samples containing NBD-111p-91 integration intermediates prepared with (a) KRMs, (b) XRMs, (c) XRMs + αSec61α, or (d) RRMs (XRMs reconstituted with rBiP) were divided into four equal aliquots. Each sample then received the same total concentration of KCl and KI, but different amounts of KI as shown. Fo is the net fluorescence intensity in the absence of quencher, whereas F is the net fluorescence intensity at a given iodide ion concentration. Measurements were made in the absence (▴) or the presence (•) of pore-forming PFO toxin to introduce iodide ions into the lumen of the microsomes. The linear least-squares best-fit lines for data averaged from several independent experiments are shown. The results are also reported in Tables I and II.
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fig3: Iodide ion quenching of NBD-labeled 111p-91 integration intermediates prepared with various ER microsomes. Samples containing NBD-111p-91 integration intermediates prepared with (a) KRMs, (b) XRMs, (c) XRMs + αSec61α, or (d) RRMs (XRMs reconstituted with rBiP) were divided into four equal aliquots. Each sample then received the same total concentration of KCl and KI, but different amounts of KI as shown. Fo is the net fluorescence intensity in the absence of quencher, whereas F is the net fluorescence intensity at a given iodide ion concentration. Measurements were made in the absence (▴) or the presence (•) of pore-forming PFO toxin to introduce iodide ions into the lumen of the microsomes. The linear least-squares best-fit lines for data averaged from several independent experiments are shown. The results are also reported in Tables I and II.

Mentions: Liao et al. (1997) identified a stage in the integration process where the nascent chain was not accessible from either the cytoplasmic or the lumenal side of the ER membrane. When fluorescent integration intermediates containing an NBD-labeled, 91-residue 111p nascent chain (NBD-111p-91) and intact ER microsomes (KRM) were exposed to iodide ions, little collisional quenching of NBD fluorescence was observed (Ksv = 0.5 M−1) (Fig. 3 a; Table I). Because the Ksv is ∼2.5 M−1 when the same translation intermediate is not bound to the ER membrane (Liao et al., 1997), it is clear that the formation of the integration intermediate prevents iodide ion access to most nascent chain probes. As we have shown previously, residual quenching results from a combination of effects, each of which exposes a small number of NBD-labeled nascent chains to the cytoplasm. Some properly targeted integration intermediates dissociate from the translocon during the long spectroscopic measurements (Crowley et al., 1994), some nontargeted nascent chains adsorb to the outer microsomal membrane (this effect is worse with membrane proteins) (Hamman et al., 1997), and some ribosomal complexes are purified with the microsomes as part of a polysome (Hamman et al., 1997). Limited protease and ribonuclease treatment of the microsomes can eliminate nearly all of the NBD dyes exposed to (quenched by) cytoplasmic iodide ions and hence reduce the initial Ksv values to near 0 (Hamman et al., 1997). However, we have chosen here to avoid any risk of damaging the samples and focus instead on the difference, if any, in quenching between cytoplasmic and lumenal iodide ions (see below).


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)

Iodide ion quenching of NBD-labeled 111p-91 integration intermediates prepared with various ER microsomes. Samples containing NBD-111p-91 integration intermediates prepared with (a) KRMs, (b) XRMs, (c) XRMs + αSec61α, or (d) RRMs (XRMs reconstituted with rBiP) were divided into four equal aliquots. Each sample then received the same total concentration of KCl and KI, but different amounts of KI as shown. Fo is the net fluorescence intensity in the absence of quencher, whereas F is the net fluorescence intensity at a given iodide ion concentration. Measurements were made in the absence (▴) or the presence (•) of pore-forming PFO toxin to introduce iodide ions into the lumen of the microsomes. The linear least-squares best-fit lines for data averaged from several independent experiments are shown. The results are also reported in Tables I and II.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199230&req=5

fig3: Iodide ion quenching of NBD-labeled 111p-91 integration intermediates prepared with various ER microsomes. Samples containing NBD-111p-91 integration intermediates prepared with (a) KRMs, (b) XRMs, (c) XRMs + αSec61α, or (d) RRMs (XRMs reconstituted with rBiP) were divided into four equal aliquots. Each sample then received the same total concentration of KCl and KI, but different amounts of KI as shown. Fo is the net fluorescence intensity in the absence of quencher, whereas F is the net fluorescence intensity at a given iodide ion concentration. Measurements were made in the absence (▴) or the presence (•) of pore-forming PFO toxin to introduce iodide ions into the lumen of the microsomes. The linear least-squares best-fit lines for data averaged from several independent experiments are shown. The results are also reported in Tables I and II.
Mentions: Liao et al. (1997) identified a stage in the integration process where the nascent chain was not accessible from either the cytoplasmic or the lumenal side of the ER membrane. When fluorescent integration intermediates containing an NBD-labeled, 91-residue 111p nascent chain (NBD-111p-91) and intact ER microsomes (KRM) were exposed to iodide ions, little collisional quenching of NBD fluorescence was observed (Ksv = 0.5 M−1) (Fig. 3 a; Table I). Because the Ksv is ∼2.5 M−1 when the same translation intermediate is not bound to the ER membrane (Liao et al., 1997), it is clear that the formation of the integration intermediate prevents iodide ion access to most nascent chain probes. As we have shown previously, residual quenching results from a combination of effects, each of which exposes a small number of NBD-labeled nascent chains to the cytoplasm. Some properly targeted integration intermediates dissociate from the translocon during the long spectroscopic measurements (Crowley et al., 1994), some nontargeted nascent chains adsorb to the outer microsomal membrane (this effect is worse with membrane proteins) (Hamman et al., 1997), and some ribosomal complexes are purified with the microsomes as part of a polysome (Hamman et al., 1997). Limited protease and ribonuclease treatment of the microsomes can eliminate nearly all of the NBD dyes exposed to (quenched by) cytoplasmic iodide ions and hence reduce the initial Ksv values to near 0 (Hamman et al., 1997). However, we have chosen here to avoid any risk of damaging the samples and focus instead on the difference, if any, in quenching between cytoplasmic and lumenal iodide ions (see below).

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