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Dynamics and retention of misfolded proteins in native ER membranes

Nature Cell Biology volume 2, pages 288–295 (2000)Cite this article

Abstract

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG–GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG–GFP complexes at 40 °C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG–chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.

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Figure 1: Attachment of GFP to ts045 VSVG does not alter its temperature-dependent folding.
Figure 2: Both correctly folded (32 °C) and misfolded (40 °C) forms of VSVG–GFP are highly mobile in ER membranes.
Figure 3: The extent and time course of fluorescence recovery for VSVG–GFP in the ER are similar at 32 °C and 40 °C, but are decreased by ATP depletion.
Figure 4: Repetitive photobleaching of cells expressing KDELR–GFP and VSVG–GFP reveals different mechanisms for their retention in the ER.
Figure 5: Cells expressing KDELR–GFP contain more residual fluorescent structures than cells expressing VSVG–GFP after repeated photobleaching.
Figure 6: ER-localized VSVG–GFP is immobilized by tunicamycin treatment.
Figure 7: Immobilization of VSVG–GFP by tunicamycin treatment.
Figure 8: Diffusional mobility of the soluble ER protein ss–GFP is affected by inhibition of N-linked glycosylation.
Figure 9: Effects of ATP depletion, tunicamycin and dithiothreitol on mobility of ss–GFP.

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Acknowledgements

We thank B. Nichols, K. Hirschberg and J. Bonifacino for comments and suggestions and P. Walter (USCF, San Francisco, CA) for SRβ DNA. E.S. is supported by a grant (ROI GM59018-01) from the NIH.

Correspondence and requests for materials should be addressed to J.L-S.

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Authors and Affiliations

  1. Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Building 18T, National Institute of Health, Bethesda, 20892, Maryland, USA

    Sarah Nehls, Erik L. Snapp, Nelson B. Cole, Kristien J.M. Zaal, Anne K. Kenworthy, Theresa H. Roberts, John F. Presley & Jennifer Lippincott-Schwartz

  2. Gene Expression and Cell Biology Program, European Molecular Biology Laboratory, Heidelberg, Germany

    Jan Ellenberg

  3. Center for Studies in Physics and Biology, Rockefeller University, New York, New York, 10021, USA

    Eric Siggia

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Correspondence to Jennifer Lippincott-Schwartz.

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Nehls, S., Snapp, E., Cole, N. et al. Dynamics and retention of misfolded proteins in native ER membranes. Nat Cell Biol 2, 288–295 (2000). https://doi.org/10.1038/35010558

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