Date of Award
Doctor of Philosophy (PhD)
Molecular Physiology and Biophysics
Kathleen M. Trybus
mRNA localization ensures correct spatial and temporal control of protein synthesis in the cell. Using a single molecule in vitro approach, we provide insight into the mechanisms by which localizing mRNAs are carried by molecular motors on cytoskeletal tracks to their destination.
Budding yeast serves as a model system for studying the mechanisms of mRNA transport because localizing mRNAs are moved on actin tracks in the cell by a single class V myosin motor, Myo4p. Molecular motors that specialize in cargo transport are generally double-headed so that they can "walk" for many microns without dissociating, a feature known as processivity. Thus, is was surprising when Myo4p purified from yeast was shown by in vitro assays to be non-processive. The reason for its inability to move processively is that the Myo4p heavy chain does not dimerize with itself, but instead binds tightly to the adapter protein She3p to form a single-headed motor complex. The mRNA-binding adapter protein She2p links Myo4p to mRNA cargo by binding She3p.
To understand the molecular mechanisms of mRNA transport in budding yeast, we fully reconstituted a messenger ribonucleoprotein (mRNP) complex from purified proteins and a localizing mRNA (ASH1) found in budding yeast. Using single molecule in vitro assays, we find that She2p recruits two Myo4p-She3p complexes, forming a processive double-headed motor complex that is stabilized by mRNA at physiological ionic strength. Thus, only in the presence of mRNA is Myo4p capable of continuous mRNA transport, an elegant mechanism that ensures that only cargo bound motors are motile.
We next wished to understand if the principles of mRNA transport in budding yeast are conserved in higher eukaryotes. In Drosophila, mRNA is transported on microtubule tracks by cytoplasmic dynein, and the adapters that link the motor to localizing transcripts are well-defined. The adapter protein bicaudal D (BicD) coordinates dynein motor activity with mRNA cargo binding. The N-terminus of BicD binds dynein, and the C-terminus interacts with the mRNA-binding protein Egalitarian. Unlike mammalian dynein alone, it was recently shown that an N-terminal fragment of BicD (BicD2CC1), in combination with a large 1.2MDa multi-subunit accessory complex called dynactin, forms a complex (DDBCC1) that is activated for long processive runs. But unlike the constitutively activated BicD2CC1 fragment, the full-length BicD molecule fails to recruit dynein-dynactin because it is auto-inhibited by interactions between the N-terminal dynein binding domain and the C-terminal cargo binding domain. To understand how dynein is activated by native cargo and full-length adapters, we fully reconstituted a mRNP complex in vitro from tissue-purified dynein and dynactin, expressed full-length adapters BicD and Egalitarian, and a synthesized localizing mRNA found in Drosophila. We find that only mRNA-bound Egalitarian is capable of relieving BicD auto-inhibition for the recruitment of dynein-dynactin, and activation of mRNA transport in vitro. Thus, the presence of an mRNA cargo for activation of motor complexes is a conserved mechanism in both budding yeast and higher eukaryotes to ensure that motor activity is tightly coupled to cargo selection.
Number of Pages
Sladewski, Thomas Edward, "Molecular Mechanisms Of Mrna Transport By A Class V Myosin And Cytoplasmic Dynein" (2017). Graduate College Dissertations and Theses. 689.