Student Theses and Dissertations

Date of Award

2016

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Rout Laboratory

Abstract

Cellular functions are performed by the concerted action of macromolecular assemblies. These protein machines rarely have permanent, invariable structures. Instead, the subunits forming the macromolecular assemblies exchange between free and bound states, changing the composition, conformation and function of the assembly. Characterizing the function of macromolecular assemblies, therefore, requires the study of their dynamics. I have developed a strategy for in vivo assessment of macromolecular plasticity and have successfully applied it to the yeast nuclear pore complex (NPC), one of the largest and most elaborate protein machines in the cell. NPC is a massive protein assembly situated in the nuclear envelope and mediating macromolecular transport between the nucleus and cytoplasm. To study the NPC plasticity I have combined multiple methodologies: affinity capture, metabolic labeling and mass spectrometry. Moreover, to identify the required affinity capture conditions I have developed a high-throughput screen, which has successfully worked with numerous protein complexes, in addition to the NPC, to reveal novel interactions and arrangements of proteins within a complex. My study on NPC plasticity has produced the first comprehensive description of NPC subunit dynamics, although for the most dynamic subunits I can only approximately estimate the exchange rate. I have also mapped the subunit dynamics on the architecture of the NPC. The central core of the NPC was revealed to be very stable, while the peripherally associated subunits exchanged with varying rates. Moreover, the rate of exchange appeared well correlated with the strength of the interaction that the NPC subunit forms with the scaffold. Notably, NPC subunits directly involved in the transport function of the NPC were among the most dynamic ones, implying that modulating the association dynamics of those subunits might alter the nuclear transport function. The dynamics of NPC modules, as well as the plasticity changes in NPC subunit mutants have suggested that the core of the complex is extremely stable because of the additive effect of numerous weak interactions formed between NPC subunits and also the surrounding nuclear envelope membrane. The individual weak interactions may be a mechanism to prevent off target NPC assembly. Lastly, I propose a study to directly test the existence of a potential NPC repair mechanism, which is a topic of heated debate and has direct implication for aging in all eukaryotes.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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