Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins

Jared Hennen, John Kohler, Siddarth Reddy Karuka, Cosmo A. Saunders, G. W.Gant Luxton, Joachim D. Mueller

Research output: Contribution to journalArticlepeer-review

Abstract

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.

Original languageEnglish (US)
Pages (from-to)2385-2399
Number of pages15
JournalBiophysical journal
Volume118
Issue number10
DOIs
StatePublished - May 19 2020

Bibliographical note

Funding Information:
We thank Steven Vogel (National Institutes of Health) for providing plasmids for the Venus concatamers and Phyllis Hanson (University of Michigan) for the SS-torsinAWT-EGFP construct. We also thank Yan Chen (University of Minnesota) and Patrick T. Willey (University of Minnesota) for providing technical assistance. This work was supported by the National Institutes of Health GM064589 (J.D.M. J.H. J.K. and S.R.K.) and GM129374 (G.W.G.L. and J.D.M.).

Funding Information:
This work was supported by the National Institutes of Health GM064589 (J.D.M., J.H., J.K., and S.R.K.) and GM129374 (G.W.G.L. and J.D.M.).

Publisher Copyright:
© 2020 Biophysical Society

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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