Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Identification and characterization of protein-protein interactions in the nuclear envelope
Stockholm University, Faculty of Science, Department of Neurochemistry.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The nuclear envelope forms the interface between the nucleus and the cytoplasm. The nuclear envelope consists of the two concentric lipid membranes, the nuclear pores and the nuclear lamina. The inner nuclear membrane contains hundreds of unique transmembrane proteins showing high tissue diversity. Mutations of some proteins in the nuclear envelope give rise to a broad spectrum of diseases called envelopathies or laminopathies. In this thesis, I aimed to study the functional organization of the nuclear envelope by identifying and characterizing interactions between the nuclear envelope proteins. For this, we developed a novel method called the Membrane Protein Crosslink Immuno-Precipitation, which enable identification of protein-protein interactions in the nuclear envelope in live cells. We identified several novel interactions of the inner nuclear membrane protein, Samp1, and studied the interaction between the Samp1 and the nuclear GTPase, Ran in detail. Samp1 can bind to Ran and is thus the first known transmembrane Ran binding protein and Samp1 might provide a local binding site for Ran in the inner nuclear membrane. We found that Samp1 also binds to the inner nuclear membrane protein, Emerin and Ran can regulate the Samp1-Emerin interaction in the nuclear envelope. During mitosis, Samp1 distributes in the mitotic spindle. Therefore, we investigated a possible functional role of Samp1 in the mitotic machinery. Samp1 depletion resulted in aneuploid phenotypes, metaphase prolongation and decreased distribution of γ-tubulin and β-tubulin in the mitotic spindle. We found that Samp1 can bind to γ-tubulin, which is essential for the microtubule nucleation and hence for the spindle stability. The new interesting features of Samp1 provide insights on the unforeseen functions of the nuclear envelope proteins.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University , 2017.
Keyword [en]
Nuclear envelope, Samp1, Emerin, Ran and transmembrane proteins
National Category
Other Chemistry Topics
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-148432ISBN: 978-91-7797-029-3 (print)ISBN: 978-91-7797-030-9 (electronic)OAI: oai:DiVA.org:su-148432DiVA, id: diva2:1152378
Public defence
2017-12-08, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2017-11-15 Created: 2017-10-24 Last updated: 2017-11-16Bibliographically approved
List of papers
1. MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
Open this publication in new window or tab >>MCLIP, an effective method to detect interactions of transmembrane proteins of the nuclear envelope in live cells
Show others...
2014 (English)In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 10, p. 2399-2403Article in journal (Refereed) Published
Abstract [en]

Investigating interactions of proteins in the nuclear envelope (NE) using co-immunoprecipitation (Co-IP) has previously been difficult or even impossible due to their inherent resistance to extraction. We have developed a novel method, MCLIP (Membrane protein Cross-Link ImmunoPrecipitation), which takes advantage of a cell permeable crosslinker to enable effective detection and analysis of specific interactions of NE proteins in live cells using Western blot. Using MCLIP we show that, in U2OS cells, the integral inner nuclear membrane protein Samp1 interacts with Lamin B1, the LINC (Linker of nucleoskeleton and cytoskeleton) complex protein, Sun1 and the soluble small GTPase Ran. The results show that the previously detected in vitro interaction between Samp1 and Emerin also takes place in live cells. In vitro pull down experiments show, that the nucleoplasmic domains of Samp1 and Emerin can bind directly to each other. We also, show that MCLIP is suitable to coprecipitate protein interactions in different stages of the cell cycle.

Keyword
Samp1, Nuclear envelope, Nuclear membrane, Crosslinking, CoIP, Protein–protein interaction
National Category
Chemical Sciences Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-109181 (URN)10.1016/j.bbamem.2014.06.008 (DOI)000340975600005 ()
Funder
Swedish Research Council, 621-2010-448Swedish Cancer Society, 110590
Available from: 2014-11-14 Created: 2014-11-14 Last updated: 2018-03-20Bibliographically approved
2. Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane
Open this publication in new window or tab >>Samp1, a RanGTP binding transmembrane protein in the inner nuclear membrane
2016 (English)In: Nucleus, ISSN 1949-1034, E-ISSN 1949-1042, Vol. 7, no 4, p. 415-423Article in journal (Refereed) Published
Abstract [en]

Samp1 is a transmembrane protein of the inner nuclear membrane (INM), which interacts with the nuclear lamina and the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex in interphase and during mitosis, it localizes to the mitotic spindle. Samp1 was recently found to coprecipitate a protein complex containing Ran, a GTPase with fundamental regulatory functions both in interphase and in mitosis. To investigate the interaction between Samp1 and Ran in further detail, we have designed and expressed recombinant fusion proteins of the Chaetomium thermophilum homolog of Samp1 (Ct. Samp1) and human Ran. Pulldown experiments show that Samp1 binds directly to Ran and that Samp1 binds better to RanGTP compared to RanGDP. Samp1 also preferred RanGTP over RanGDP in living tsBN2 cells. We also show that the Ran binding domain is located between amino acids 75-135 in the nucleoplasmically exposed N-terminal tail of Samp1. This domain is unique for Samp1, without homology in any other proteins in fungi or metazoa. Samp1 is the first known transmembrane protein that binds to Ran and could provide a unique local binding site for RanGTP in the INM. Samp1 overexpression resulted in increased Ran concentrations in the nuclear periphery supporting this idea.

Keyword
EDMD, laminopathies, LINC complex, nucleus, nuclear membrane, Ran
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-135087 (URN)10.1080/19491034.2016.1220465 (DOI)000384442800010 ()27541860 (PubMedID)
Available from: 2016-11-23 Created: 2016-10-31 Last updated: 2017-10-30Bibliographically approved
3. RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin
Open this publication in new window or tab >>RanGTPase regulates the interaction between the inner nuclear membrane proteins, Samp1 and Emerin
Show others...
(English)Manuscript (preprint) (Other (popular science, discussion, etc.))
National Category
Biological Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-148431 (URN)
Available from: 2017-10-24 Created: 2017-10-24 Last updated: 2017-10-30Bibliographically approved
4. Mitotic spindle assembly and correct chromosome segregation depend on the integral nuclear membrane protein, Samp1
Open this publication in new window or tab >>Mitotic spindle assembly and correct chromosome segregation depend on the integral nuclear membrane protein, Samp1
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-141814 (URN)
Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2017-10-30Bibliographically approved

Open Access in DiVA

Identification and characterization of protein-protein interactions in the nuclear envelope(959 kB)79 downloads
File information
File name FULLTEXT01.pdfFile size 959 kBChecksum SHA-512
c80a8ac27c13e6d7d6c121a42e31b964ca39ecb02732cba2bcbae558317b060d6075513ce291deb9f5deecb81228fc2a1ab75c3a44d1574f866458c1e503bc7d
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Vijayaraghavan, Balaje
By organisation
Department of Neurochemistry
Other Chemistry Topics

Search outside of DiVA

GoogleGoogle Scholar
Total: 79 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 1431 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf