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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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Three possible mechanisms for sealing the lumenal side of the aqueous translocon pore during protein integration. (a) A conformational change in the translocon prevents ion flow through the pore from the lumenal end. (b) Disassembly of the translocon machinery maintains the permeability barrier of the membrane by eliminating the pore. (c) A soluble lumenal protein mediates, either directly or indirectly, the closure of the lumenal side of the aqueous translocon pore.
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fig1: Three possible mechanisms for sealing the lumenal side of the aqueous translocon pore during protein integration. (a) A conformational change in the translocon prevents ion flow through the pore from the lumenal end. (b) Disassembly of the translocon machinery maintains the permeability barrier of the membrane by eliminating the pore. (c) A soluble lumenal protein mediates, either directly or indirectly, the closure of the lumenal side of the aqueous translocon pore.

Mentions: By what mechanism is the lumenal end of the translocon pore closed or gated during membrane protein integration? One possibility is that a conformational change in the translocon narrows the pore and thereby prevents ion flow (Fig. 1 a). Because the translocon has been shown to undergo structural changes that greatly alter pore diameter (Hamman et al., 1997, 1998), this is a likely possibility. Alternatively, the translocon may disassemble so that the pore disappears (Fig. 1 b). We consider this an unlikely scenario because the translocon pore remains intact even when no ribosome is bound to the translocon (Hamman et al., 1998). A third possibility is that a soluble lumenal protein effects, directly or indirectly, closure of the pore (Fig. 1 c). Consistent with such a scenario, BiP, a lumenal Hsp70, is responsible for closing the translocon pore before ribosome binding and for a short time after the ribosome is targeted to the translocon (Crowley et al., 1994; Hamman et al., 1998). However, a translocon at rest and a translocon engaged in integration differ structurally because the latter is bound to a ribosome and also has a nascent chain extending through the translocon (Liao et al., 1997). If some lumenal protein closes the pore during integration as depicted in Fig. 1c, the presence of the nascent chain makes the closure mechanism substantially more complicated than that of BiP closing a ribosome-free translocon pore that lacks a nascent chain. An even greater complication is the fact that not all pores occupied by a nascent chain will be closed: those pores translocating secretory proteins will not be closed (Crowley et al., 1994), and only some pores occupied by a nascent membrane protein will be closed at any given time (Liao et al., 1997). Thus, irrespective of the mechanism of pore closure, a sophisticated regulatory mechanism must have evolved for identifying which pores containing nascent chains need to be closed. In this study, a fluorescence quenching approach was used to examine these important and fundamental issues and to distinguish between the above three possibilities.


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)

Three possible mechanisms for sealing the lumenal side of the aqueous translocon pore during protein integration. (a) A conformational change in the translocon prevents ion flow through the pore from the lumenal end. (b) Disassembly of the translocon machinery maintains the permeability barrier of the membrane by eliminating the pore. (c) A soluble lumenal protein mediates, either directly or indirectly, the closure of the lumenal side of the aqueous translocon pore.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Three possible mechanisms for sealing the lumenal side of the aqueous translocon pore during protein integration. (a) A conformational change in the translocon prevents ion flow through the pore from the lumenal end. (b) Disassembly of the translocon machinery maintains the permeability barrier of the membrane by eliminating the pore. (c) A soluble lumenal protein mediates, either directly or indirectly, the closure of the lumenal side of the aqueous translocon pore.
Mentions: By what mechanism is the lumenal end of the translocon pore closed or gated during membrane protein integration? One possibility is that a conformational change in the translocon narrows the pore and thereby prevents ion flow (Fig. 1 a). Because the translocon has been shown to undergo structural changes that greatly alter pore diameter (Hamman et al., 1997, 1998), this is a likely possibility. Alternatively, the translocon may disassemble so that the pore disappears (Fig. 1 b). We consider this an unlikely scenario because the translocon pore remains intact even when no ribosome is bound to the translocon (Hamman et al., 1998). A third possibility is that a soluble lumenal protein effects, directly or indirectly, closure of the pore (Fig. 1 c). Consistent with such a scenario, BiP, a lumenal Hsp70, is responsible for closing the translocon pore before ribosome binding and for a short time after the ribosome is targeted to the translocon (Crowley et al., 1994; Hamman et al., 1998). However, a translocon at rest and a translocon engaged in integration differ structurally because the latter is bound to a ribosome and also has a nascent chain extending through the translocon (Liao et al., 1997). If some lumenal protein closes the pore during integration as depicted in Fig. 1c, the presence of the nascent chain makes the closure mechanism substantially more complicated than that of BiP closing a ribosome-free translocon pore that lacks a nascent chain. An even greater complication is the fact that not all pores occupied by a nascent chain will be closed: those pores translocating secretory proteins will not be closed (Crowley et al., 1994), and only some pores occupied by a nascent membrane protein will be closed at any given time (Liao et al., 1997). Thus, irrespective of the mechanism of pore closure, a sophisticated regulatory mechanism must have evolved for identifying which pores containing nascent chains need to be closed. In this study, a fluorescence quenching approach was used to examine these important and fundamental issues and to distinguish between the above three possibilities.

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