GSBS Dissertations and Theses

Approval Date

July 1992

Document Type

Doctoral Dissertation

Department

Graduate School of Biomedical Sciences, Cell Biology

Subjects

Cell Movement; Academic Dissertations; Dissertations, UMMS

Abstract

In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes.

The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase.

The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas 70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes.

The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.

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