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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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111p protein and integration intermediates. (a) The chimeric 111p single-spanning membrane protein was constructed using the TM sequence from vesicular stomatitus G protein (residues 65–84, dark gray) and lysine-free stretches of preprolactin and Bcl-2 (Liao et al., 1997). 111p contains only a single lysine codon at position 75 in the middle of the TM sequence (white circle). Note that the TM segment will still be nonpolar and uncharged when ɛNBD-Lys is incorporated at this location. The signal sequence (SS) is indicated in gray (residues 1–22), and the triangle indicates the approximate position of the truncations used in this study (residues 86, 91, and 93). (b) Accessibility of the fluorescent probe from either the cytoplasmic or lumenal sides of intact ER microsomes is indicated for each intermediate used in this study (Liao et al., 1997). The ribosome is shown bound to the translocon (black) at the ER membrane (light gray) in each case. The TM sequence is represented by a dark gray rectangle and the fluorescent probe by a white circle.
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fig2: 111p protein and integration intermediates. (a) The chimeric 111p single-spanning membrane protein was constructed using the TM sequence from vesicular stomatitus G protein (residues 65–84, dark gray) and lysine-free stretches of preprolactin and Bcl-2 (Liao et al., 1997). 111p contains only a single lysine codon at position 75 in the middle of the TM sequence (white circle). Note that the TM segment will still be nonpolar and uncharged when ɛNBD-Lys is incorporated at this location. The signal sequence (SS) is indicated in gray (residues 1–22), and the triangle indicates the approximate position of the truncations used in this study (residues 86, 91, and 93). (b) Accessibility of the fluorescent probe from either the cytoplasmic or lumenal sides of intact ER microsomes is indicated for each intermediate used in this study (Liao et al., 1997). The ribosome is shown bound to the translocon (black) at the ER membrane (light gray) in each case. The TM sequence is represented by a dark gray rectangle and the fluorescent probe by a white circle.

Mentions: A chimeric single-spanning membrane protein designated 111p that has been well characterized previously (Liao et al., 1997) was used in this study (Fig. 2 a). The 111p coding sequence contains only a single lysine codon that positions a fluorescent probe in the center of the nascent chain TM sequence in each integration intermediate. At the lengths of nascent chain analyzed in this study, the TM sequence, and therefore the fluorescent probe, are located in the ribosomal nascent chain tunnel in each integration intermediate (Fig. 2 b).


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)

111p protein and integration intermediates. (a) The chimeric 111p single-spanning membrane protein was constructed using the TM sequence from vesicular stomatitus G protein (residues 65–84, dark gray) and lysine-free stretches of preprolactin and Bcl-2 (Liao et al., 1997). 111p contains only a single lysine codon at position 75 in the middle of the TM sequence (white circle). Note that the TM segment will still be nonpolar and uncharged when ɛNBD-Lys is incorporated at this location. The signal sequence (SS) is indicated in gray (residues 1–22), and the triangle indicates the approximate position of the truncations used in this study (residues 86, 91, and 93). (b) Accessibility of the fluorescent probe from either the cytoplasmic or lumenal sides of intact ER microsomes is indicated for each intermediate used in this study (Liao et al., 1997). The ribosome is shown bound to the translocon (black) at the ER membrane (light gray) in each case. The TM sequence is represented by a dark gray rectangle and the fluorescent probe by a white circle.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199230&req=5

fig2: 111p protein and integration intermediates. (a) The chimeric 111p single-spanning membrane protein was constructed using the TM sequence from vesicular stomatitus G protein (residues 65–84, dark gray) and lysine-free stretches of preprolactin and Bcl-2 (Liao et al., 1997). 111p contains only a single lysine codon at position 75 in the middle of the TM sequence (white circle). Note that the TM segment will still be nonpolar and uncharged when ɛNBD-Lys is incorporated at this location. The signal sequence (SS) is indicated in gray (residues 1–22), and the triangle indicates the approximate position of the truncations used in this study (residues 86, 91, and 93). (b) Accessibility of the fluorescent probe from either the cytoplasmic or lumenal sides of intact ER microsomes is indicated for each intermediate used in this study (Liao et al., 1997). The ribosome is shown bound to the translocon (black) at the ER membrane (light gray) in each case. The TM sequence is represented by a dark gray rectangle and the fluorescent probe by a white circle.
Mentions: A chimeric single-spanning membrane protein designated 111p that has been well characterized previously (Liao et al., 1997) was used in this study (Fig. 2 a). The 111p coding sequence contains only a single lysine codon that positions a fluorescent probe in the center of the nascent chain TM sequence in each integration intermediate. At the lengths of nascent chain analyzed in this study, the TM sequence, and therefore the fluorescent probe, are located in the ribosomal nascent chain tunnel in each integration intermediate (Fig. 2 b).

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