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Cells 2015, 4(4), 653-673; doi:10.3390/cells4040653

Active Nuclear Import of Membrane Proteins Revisited

1
European Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, Netherlands Proteomics Centre, Antonius Deusinglaan 1, Groningen 9713 AV, The Netherlands
2
Department of Biochemistry, University of Groningen, Nijenborgh 4, Groningen 9747 AG, The Netherlands
3
Skolkovo Institute of Science and Technology (Skoltech), Novaya St. 100, Skolkovo 143025, Russia
These authors contributed equally to this work.
*
Author to whom correspondence should be addressed.
Academic Editor: Birthe Fahrenkrog
Received: 10 July 2015 / Revised: 14 September 2015 / Accepted: 30 September 2015 / Published: 13 October 2015
(This article belongs to the Special Issue Nucleocytoplasmic Transport)
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Abstract

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. View Full-Text
Keywords: nuclear pore complex; inner nuclear membrane; nuclear envelope; nuclear transport nuclear pore complex; inner nuclear membrane; nuclear envelope; nuclear transport
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Laba, J.K.; Steen, A.; Popken, P.; Chernova, A.; Poolman, B.; Veenhoff, L.M. Active Nuclear Import of Membrane Proteins Revisited. Cells 2015, 4, 653-673.

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