The nucleus of eukaryotic cells houses the genetic material (DNA) that encodes the blueprint of life. The nuclear envelope (NE), the membrane system that bounds the nucleus, is a double membrane with outer and inner membranes. The outer nuclear membrane (ONM), which faces the cytoplasm, is continuous with and biochemically similar to the endoplasmic reticulum (ER), the site of synthesis for nearly all integral membrane and secreted proteins. The inner nuclear membrane (INM) contacts the nucleoplasm and contains a specific set of integral membrane proteins that participate in a variety of important processes including maintenance of nuclear architecture, chromatin organization, signaling and gene expression.

Findings that significantly advance the understanding of how the integrity of the INM is maintained are published  in NATURE on December 18, 2014. The underlying research utilized the yeast Saccharomyces cerevisiae as a model system and was carried out by an international team of experimentalists in Canada, France, Germany and Sweden. The team discovered a novel quality control pathway associated with the INM. The publication documents that the Asi1/Asi3 complex, comprised of two integral INM proteins with RING domains, function as an E3-ubiquitin ligase. Together with E2-ubiquitin conjugating enzymes Ubc6 and Ubc7 the Asi1/Asi3-complex plays an essential role in clearing the nuclear compartment from mislocalized soluble and integral membrane proteins; ubiquitylated proteins are targeted to proteasomes for degradation.

“Ever since we discovered the Asi proteins, a little more than 13 years ago (Forsberg et al. 2001), and found that they have RING domains, we predicted that they would have a role in protein quality control. But it has taken us until early this year to obtain any real evidence for this (Omnus and Ljungdahl, 2014). The wonderful collaboration that led to the publication in NATURE provides a precise molecular description of the INM associated degradation, or INMAD, pathway and, importantly, put the Asi proteins in broader biological context as a major safeguard of the integrity of the nucleus, says Professor Per O. Ljungdahl from the Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University. Prof. Ljungdahl continues, "One of the most interesting findings regarding the newly discovered INMAD quality control pathway is that many membrane proteins that should primarily localize elsewhere slip past the barrier function of nuclear pore complexes and mislocalize to the INM. A finding that explains the need for a dedicated quality control system associated with this important membrane. Also based on its unique location, the INMAD machinery is clearly distinct from the previously characterized ER-associated degradation (ERAD) pathways." Using an entirely different approach, Fortesti et al. derived quite similar conclusions in a paper recently published in SCIENCE. Together the new results point to the importance of quality control mechanisms that function to correct errors inherent to the intracellular transport of proteins, such as the removal of proteins mislocalized to the nucleus due to their accidental transport past nuclear pores. Apparently, the INMAD pathway provides cells with a solution to the inherently difficult challenge of creating fail-safe targeting mechanisms to ensure the proper composition of the nuclear compartment.

The novel conclusions published in NATURE are based on a three-pronged experimental initiative combining two state-of-the-art technical approaches and genetic and biochemical assays. First, at the Université de Rennes, researchers applied a directed bimolecular fluorescence complementation (BiFC) assay to screen for proteins that interact with E2-ubiquitin-conjugating enzymes Ubc6 and Ubc7. This identified the INM Asi1/Asi3-complex as a clear and specific interaction partner. Second, at Stockholm University, researchers used genetic tests and biochemical assays to show that the Asi1/Asi3-complex functions as an E3-ubiquitin ligase that catalyzes the in vivo polyubiquitylation of Stp1 and Stp2 when these transcription factors inappropriately enter the nucleus. Third, the research team working in Heidelberg developed a novel yeast resource, the tTF-protein timer strain library, to carry out genome-wide screening of changes in protein stability compatible with high-throughput approaches uniquely available at the University of Toronto. The results from this genome-wide assay led to the unbiased identification of a large range of mislocalized membrane protein substrates for the INM Asi-E3 ubiquitin ligase. The successful use of tTF -library represents a major technical advance that will have a significant impact in the field of protein degradation and protein homeostasis research.

"Although it is presently unclear whether similar INMAD quality control pathways exist outside of yeast, our findings will hopefully encourage other researchers to pursue the possibility that functional INMAD pathways exist and operate analogously in other organisms. Clearly safeguarding the integrity of the INM must be a critical and common concern shared by most, if not all eukaryotic cells. The importance of membrane-associated protein degradation mechanisms and the large diversity of integral membrane RING domain proteins in mammalian cells suggest that such research will lead to fruitful findings,” notes Per Ljungdahl.

 

NATURE publication:

Protein quality control at the inner nuclear membrane (2014) Khmelinskii1*, A., Blaszczak2,3*, E., Pantazopoulou4, M., Fischer5,6, B., Omnus4, D.J., Le Dez2,3, G., Brossard2,3, A., Gunnarsson4, A., Barry5, J.D., Meurer1, M., Kirrmaier1, D., Boone7, C., Huber5, W., Rabut2,3, G., Ljungdahl4, P.O., and Knop1,8, M. NATURE 516, 410-413.

 

1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

2Centre National de la Recherche Scientifique, UMR 6290, 35000 Rennes, France.

3Institut de Génétique et Développement de Rennes, Universite´ de Rennes 1, 35000 Rennes, France.

4Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Svante Arrhenius väg 20B, SE-106 91 Stockholm, Sweden.

5Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstraße 1, 69117 Heidelberg, Germany.

6Computational Genome Biology, German Cancer Research Center (DKFZ), ImNeuenheimer Feld 580, 69120 Heidelberg, Germany.

7Department of Molecular Genetics, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College St, Toronto, Ontario M5S3E1, Canada.

8Cell Morphogenesis and Signal Transduction, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

*These authors contributed equally to this work.

Additional References:

Omnus, D.J. & Ljungdahl, P.O. (2014) Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron.  Mol. Biol. Cell 25, 3823-3833.

Forsberg, H., Hammar, M., Andréasson, C., Moliner, A. & Ljungdahl, P.O. (2001) Suppressors of ssy1 and ptr3 Null Mutations Define Novel Amino Acid Sensor Independent (ASI) genes in Saccharomyces cerevisiae. Genetics 158, 973-988.

Foresti, O., Rodriguez-Vaello, V., Funaya, C. & Carvalho, P. (2014) Quality control of inner nuclear membrane proteins by the Asi complex. Science 346, 751–755.

For more information:

Per O. Ljungdahl, Professor, Department of Molecular Biosciences, The Wenner-Gren Institute (MBW), tfn +46 8 16 41 01, e-mail: per.ljungdahl@su.se

The study was funded in part by the Swedish Research Council.

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