MITOTIC REGULATION OF THE NUCLEAR PORE COMPLEX PROTEIN INTERACTION NETWORK
EMSL Project ID
24591
Abstract
Eukaryotic cells segregate DNA equally to daughter cells during mitosis. Although mitosis is well characterized, it is poorly understood at the molecular level. The most extensive and least understood mitotic process is nuclear breakdown. Because nuclear breakdown does not occur in model yeasts it cannot be studied using these powerful model systems. However, my lab has discovered that in the model filamentous fungus Aspergillus nidulans certain aspects of nuclear breakdown occurs during mitosis. Most importantly, A. nidulans disassembles its nuclear pore complex (NPC) as do human cells that undergo mitotic nuclear breakdown (open mitosis). This finding has provided new concepts regarding how closed mitosis is regulated and is the basis for this application.The NPC is a huge, dynamic, multiprotein complex spanning the nuclear envelope. It provides the regulated conduit between the nucleoplasm and cytoplasm. It is composed of multiple copies of 25-30 conserved proteins which interact to form NPC subcomplexes, the building blocks of the NPC. Although during open mitosis stepwise disassembly then reassembly of the NPC occurs, how this is regulated is not known. In A. nidulans, over half of the NPC proteins (14/26) disperse from the NPC at mitosis before returning to the core NPC structure during mitotic exit. We wish to understand how these massive changes are regulated and want to define the molecular mechanisms involved.
A. nidulans provides nearly all of the advantages of working with the most powerful model eukaryote system, Saccharomyces cerevisiae. Its genome has been sequenced and highly efficient gene manipulations by targeted gene replacements are routine. Importantly, we have recently affinity purified to homogeneity several endogenously tagged NPC proteins. This has identified 5 NPC subcomplexes. Using mass spectroscopy we have identified 11 proteins in these subcomplexes, one of which was found to be phosphorylated during mitosis.
In this application we wish to define, in a systematic manner, how the protein-protein interactions within the NPC change from interphase to mitosis. Secondly, we want to establish which subunits of the NPC are modified during mitosis. All NPC proteins of A. nidulans are to be individually purified from both interphase and mitotic cell extracts. This will be done using established techniques and the S-Tag affinity purification system. Protein complexes will be released from the affinity matrix using NH4OH. We then wish to utilize the MS facilities at EMSL to identify the constituents of the purifications and define the post translational modifications, particularly mitotic specific phosphorylations, of the purified proteins.
Using this systematic approach a roadmap of the dynamic protein interaction networks of the NPC will be generated. This will advance our understanding of the ways by which a massive protein structure can be dismantled and reassembled and set the stage to define the regulatory mechanisms involved. The findings will have a major impact on our understanding of the mitotic regulation of the NPC and will be published in the top scientific journals. The proposed research fits well with the EMSL Science Theme of determining the dynamics of protein composition or localization and their assembly into multiprotein complexes.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-05-25
End Date
2010-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Zhao R, SJ Ding, Y Shen, DG Camp, II, EA Livesay, HR Udseth, and RD Smith. 2009. "Automated metal-free multiple-column nanoLC for improved phosphopeptide analysis sensitivity and throughput." Journal of Chromatography B 877(8-9):663-670.