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Nora G. Haigh¹ and Arthur E. Johnson^1,2,3

¹ Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center, College Station, TX 77843
² Department of Chemistry, Texas A&M University, College Station, TX 77843
³ Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843

Address correspondence to Arthur E. Johnson, College of Medicine, Texas A&M University System Health Science Center, 116 Reynolds Medical Bldg., TAMUS HSC, 1114 TAMU, College Station, TX 77843-1114. Tel.: (979) 862-3188. Fax: (979) 862-3339. E-mail: aejohnson{at}tamu.edu

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.

Key Words: BiP; protein integration; endoplasmic reticulum; translocon; fluorescence spectroscopy

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