Abstract
AbstractSUN proteins are an integral part of LINC (Linker of Nucleoskeleton and Cytoskeleton) complex which spans the nuclear envelope and acts as a physical tether between the cytoskeletal filaments and the nuclear lamina. Several human diseases associated with nuclear deformation are primarily caused by impaired functioning of SUN proteins. Studies in yeast and mammalian cells have illustrated the detrimental effects of different SUN mutants in nuclear positioning and movement. While cell-based studies provide physiological relevance to the functioning of a protein, in vitro reconstitution of isolated proteins is useful in mechanistically dissecting protein function in a biochemically defined environment. In this study, we used a mammalian cell-free expression system to synthesize and reconstitute SUN proteins in artificial lipid bilayer membranes. Building on our previous work demonstrating directional reconstitution of full-length SUN proteins, we deciphered the mechanism of such protein reconstitution and leveraged it to test several theories/models of LINC complex assembly. By using a simple fluorescence-based assay, we revealed the importance of cations such as calcium and the presence of disulfide bonds in the formation of LINC complexes. Through sequential reconstitutions of SUN proteins and soluble luminal domains of SUN proteins, we found that coiled coil domains of SUN proteins are necessary for homomeric and heteromeric interactions of reconstituted SUN proteins. Overall, our results provide mechanistic insights on LINC complex formation and how this might impact cellular mechanotransduction. The facile approach for reconstituting full-length membrane proteins can be extended to investigate other difficult-to-study membrane proteins in vitro.
Publisher
Cold Spring Harbor Laboratory
Reference48 articles.
1. M. Crisp , et al., Coupling of the nucleus and cytoplasm: Role of the LINC complex. J. Cell Biol. (2006) https:/doi.org/10.1083/jcb.200509124.
2. B. A. Sosa , U. Kutay , T. U. Schwartz , Structural insights into LINC complexes. Curr. Opin. Struct. Biol. 23 (2013).
3. B. A. Sosa , A. Rothballer , U. Kutay , T. U. Schwartz , LINC Complexes Form by Binding of Three KASH Peptides to Domain Interfaces of Trimeric SUN Proteins. Cell 149 (2012).
4. C. Ostlund , et al., Dynamics and molecular interactions of linker of nucleoskeleton and cytoskeleton (LINC) complex proteins. J. Cell Sci. 122 (2009).
5. Q. Wang , X. Du , Z. Cai , M. I. Greene , Characterization of the Structures Involved in Localization of the SUN Proteins to the Nuclear Envelope and the Centrosome. DNA Cell Biol. 25 (2006).
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