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Identification of Dynein Binding Sites in Budding Yeast Pac1/LIS1

Abstract
Pac1/LIS1, an essential tip tracking protein of the WD40 super family, is required to target cytoplasmic dynein to the plus ends of astral microtubules in budding yeast. Pac1/LIS1 protein is composed of two regions: a small coiled-coil domain and a highly conserved WD40 repeat domain. Because of in vivo data suggesting the motor domain of Dyn1 interacts with Pac1, I attempted to locate the region of Pac1/LIS1 essential for binding to Dyn1/HC by utilizing PCR-mediated site directed mutagenesis. PCR-generated site directed Pac1(S226P) mutant appears to bind Dyn1/HC, allowing it to localize to the microtubule plus ends; whereas, Pac1(H197R) and Pac1(D379H) mutants appear to disrupt motor localization. I further hypothesized that Dyn1/HC would bind to either the coiled-coil domain or the WD40 repeat domain. Using truncated Pac1 constructs, I have observed that neither the coiled-coil domain nor the WD40 repeat domain alone is sufficient to recruit Dyn1/DHC to the plus ends of the cytoplasmic microtubules. Additionally, if I dimerize the WD40 repeat domain with a GST fusion tag, I observed that Dyn1/HC colocalized with the truncation at the spindle pole bodies. This result indicates that Pac1 must dimerize with its coiled-coil domain prior to interacting with Dyn1/HC. Furthermore, the WD40 dimer, is unable to track microtubule plus-ends; indicating that the very N-terminus of Pac1 is important for other interactions responsible for recruiting the Pac1/Dyn1 complex to the astral microtubule plus end.
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2010-01-01
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