%0 Generic
%A Langer, Julian David
%D 2008
%F heidok:8708
%K Coatomer , FRET , Electron microscopy , single-molecule spectroscopy
%R 10.11588/heidok.00008708
%T Conformational dynamics of coatomer : functional and structural studies
%U https://archiv.ub.uni-heidelberg.de/volltextserver/8708/
%X In my PhD thesis I have investigated molecular mechanisms in the biogenesis of membrane vesicles. Formation of transport vesicles involves polymerization of cytoplasmic coat proteins. In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of coatomer's subunit gamma-COP of the Golgi membrane protein p23 induces a conformational change in the gamma-subunit, leading to polymerization of the complex in vitro. Using a combination of biochemical assays and an assay based on single-molecule, singlepair fluorescence resonance energy transfer, we find that this conformational change is only induced by dimers of the p24-family proteins p23 and p24, and neither by the other p24-family members nor by cargo proteins. This conformational change takes place in individual coatomer complexes, independent of each other, and the rearrangement induced in gamma-COP is transmitted within the complex to its alpha-subunit. Alpha-COP is one of coatomer's subunits capable of binding to dibasic cargo motifs, and also shows analogy to the Clathrin molecule. We propose a model in which capture of membrane protein machinery triggers cage formation in the COPI system. At the nanometer resolution I started investigating the structure of the lattice of conformationally changed coatomer on COPI vesicles generated in vitro from purified Golgi membranes and coating machinery, using cryo electron tomography. Initial data on coated vesicles and coated buds is presented.