Date of Award

Winter 2015

Project Type


Program or Major


Degree Name

Doctor of Philosophy

First Advisor

Clyde Denis

Second Advisor

Thomas Laue

Third Advisor

Subhash Minocha


Using the novel technique of analytical ultracentrifugation with fluorescent detection (AU-FDS), I have conducted the analysis of the properties of two types of intracellular macromolecular complexes: the translational mRNP complex and the intermediate soluble aggregates present in Huntington’s disease. With AU-FDS it is possible to differentiate a broad size range of soluble molecules from complex mixtures and determine the size and abundance of each individual complex based on its sedimentation rate under a centrifugal field.

In the first part of my thesis, the characteristics of the translational repressor SBP1 was determined by analyzing the mRNP complexes it was associated with. SBP1, an RNA binding protein, plays a role in stress granules and P-body function which are involved in mRNA degradation processes. SBP1, whose role in translation is unknown, was found to co-immunoprecipitate the 77S monosomal translating mRNP complex. Two types of 77S complexes containing SBP1 were identified. The majority of the 77S complexes lacked other translation initiation factors, suggesting that this 77S is in the late elongation/early termination phase. SBP1 was also present in a 77S complex that contained other initiation factors but may not be active in translation, possibly as it is located in P-bodies or stress granules.

In the second part of this thesis, the ability of the huntingtin protein, HTT-103Q, to form soluble aggregates was analyzed. HTT-103Q is a glutamine rich protein fragment previously identified as a self-propagating protein capable of forming insoluble amyloids and to cause Huntington’s disease. AU-FDS analysis identified soluble aggregates of HTT-103Q. A series of intermediate aggregates with sizes in between 30S to 180S were observed that dramatically changed in their abundance as a function of time. The deletion of chaperones HSP70 or HSP104 strongly suppressed the presence of these complexes and correspondingly suppressed amyloid formation and cell toxicity. In contrast, overexpression of the chaperones had less effect on the formation of these soluble aggregates. I also studied the effects on HTT-103Q aggregation of two aging factors known to be involved in amyloid aggregation. Overexpression of HSF1 and deletion of SIR2 significantly impaired the production of the HTT-103Q soluble aggregates. Overall, these results indicate that the 30-180S soluble aggregates detected for HTT-103Q may be critical to amyloid formation and cell toxicity.

These combined studies indicate that AU-FDS can identify and characterize novel macromolecular complexes in biological systems.