Transport of tail-anchored proteins to the inner nuclear membrane

• Applicant: Professor Dr. Ralph Kehlenbach
• Subject Area: Cell Biology
• Term: from 2013 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 234233342

The nucleus is surrounded by the inner and the outer nuclear membrane, which are connected via the nuclear pores. Rather little is known about transport of proteins to the inner nuclear membrane (INM). In one model, transmembrane proteins can passively diffuse across the nuclear pore complex. They are then sequestered at the INM upon interaction with proteins of the nuclear lamina. On the other hand, a more active transport has been described, where membrane proteins containing nuclear localization signals translocate through the nuclear pore in a complex with nuclear import receptors, similar to soluble proteins that are imported into the nucleus. Factors that are specifically required for transport of membrane proteins to the INM have not been identified so far.To investigate transport of proteins to the INM, we want to take advantage of the properties of a subset of membrane proteins, the tail-anchored proteins (TA-proteins). TA-proteins have a single transmembrane domain at their very C-terminus, requiring a post-translational mechanism for membrane insertion that is distinct from the classic, SRP/Sec61-dependent pathway. This enables us to express TA-proteins in bacteria in a complex with the chaperone TRC40, a protein that binds to the hydrophobic transmembrane domain. So far, we have used the INM-proteins emerin and LAP2 (lamina associated polypeptide 2) as model substrates. Both proteins could be post-translationally integrated into microsomal membranes, supporting the notion that they are bona fide TA-proteins. We also purified emerin tagged with the fluorophore mCherry. In digitonin-permeabilized cells, mCherry-emerin associated with the ER-membrane and the INM in a temperature-dependent manner. We are now planning to use this novel in vitro assay to reconstitute transport of emerin, LAP2 and other TA-proteins to the INM. First, we can analyze the role of nucleocytoplasmic transport factors, whose function in nuclear import of soluble proteins is well-established. At a later stage, the in vitro system will allow us to identify potential cytosolic factors that are required for efficient transport of the different cargoes. Furthermore, we will follow several approaches in intact cells and investigate the targeting of other proteins of the INM that, according to their amino acid sequence, are also substrates of the TA-protein pathway. Finally, we plan to establish a quantitative microscopy system to analyze transport of proteins to the INM in vivo and in vitro. Together, the proposed project will contribute to a better understanding of the biogenesis of the inner nuclear membrane.

• DFG Programme: Research Grants