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. 1999 Dec 27;147(7):1443–1456. doi: 10.1083/jcb.147.7.1443

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© 1999 The Rockefeller University Press

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

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graphic file with name JCB9805090.f5a.jpg — PDI specifically interacts with a misfolded, cysteine-free secretory protein. a, Wild-type PDI preferentially binds to Δgpαf. Radiolabeled pΔgpaf (lanes 1–3) or ppαf (lanes 4 and 5) were translocated into wild-type microsomes. Directly after termination of the translocation reaction, DSP cross-linking was performed as described in Materials and Methods, followed by solubilization of the membranes, and precipitation of glycoproteins with ConA-Sepharose or immunoprecipitation with anti-PDI antiserum. Cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. The positions of major cross-linking products are indicated (PDIxΔgpαf and PDIxgpαf). Note that wild-type gpαf is a glycoprotein, thus, both monomeric gpαf (•, lanes 4 and 5) and gpαf dimers (*, lane 5) bind to ConA-Sepharose, whereas Δgpαf contains no oligosaccharyl side chains and, therefore, only binds to ConA-Sepharose if cross-linked to the glycoprotein PDI (lane 2). b, Radiolabeled pΔgpαf was translocated into wild-type and pdi1 mutant microsomes and cross-linked with DSP as described. Cross-linking products of Δgpαf and wild-type or mutant PDI proteins were detected by precipitation with ConA-Sepharose.

graphic file with name JCB9805090.f5b.jpg — PDI specifically interacts with a misfolded, cysteine-free secretory protein. a, Wild-type PDI preferentially binds to Δgpαf. Radiolabeled pΔgpaf (lanes 1–3) or ppαf (lanes 4 and 5) were translocated into wild-type microsomes. Directly after termination of the translocation reaction, DSP cross-linking was performed as described in Materials and Methods, followed by solubilization of the membranes, and precipitation of glycoproteins with ConA-Sepharose or immunoprecipitation with anti-PDI antiserum. Cross-linked proteins were resolved on nonreducing 7.5% SDS gels and visualized by PhosphorImaging. The positions of major cross-linking products are indicated (PDIxΔgpαf and PDIxgpαf). Note that wild-type gpαf is a glycoprotein, thus, both monomeric gpαf (•, lanes 4 and 5) and gpαf dimers (*, lane 5) bind to ConA-Sepharose, whereas Δgpαf contains no oligosaccharyl side chains and, therefore, only binds to ConA-Sepharose if cross-linked to the glycoprotein PDI (lane 2). b, Radiolabeled pΔgpαf was translocated into wild-type and pdi1 mutant microsomes and cross-linked with DSP as described. Cross-linking products of Δgpαf and wild-type or mutant PDI proteins were detected by precipitation with ConA-Sepharose.