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Wang, Qianmin (2017): Structural and Biochemical Characterization of Cell Shaping Proteins: 1. Microtubule Binding Protein p150glued and 2. Intraflagellar Transport Protein 172. Dissertation, LMU München: Fakultät für Chemie und Pharmazie



Microtubules are cytoskeletal filaments in eukaryotic cells where they are required for cell morphogenesis, cell division and intracellular trafficking. Microtubules are highly dynamically assembled from α-/β-tubulin heterodimers. The dynamic instability of microtubules is regulated by several highly conserved microtubule associated proteins (MAPs). In particular, a spatially specialized group of MAPs that accumulate at growing microtubule ends, the plus-end binding proteins (+TIPs), is important to modulate microtubule dynamics in cells. p150glued is one of these +TIPs and is the largest subunit of the dynactin complex. Previous studies of p150glued demonstrated that it functions in recruiting and binding endosomes and dynein to microtubules for initiating retrograde transport. p150glued has two microtubule-binding domains at its N-terminus: a cytoskeleton associated proteins glycine-rich (CAP-Gly) domain, followed by a serine-rich basic domain. To understand how the p150glued CAP-Gly domain and the basic extensions interact with microtubule, cyro-electron microscopic structures of p150glued (1-105)-microtubule complex (CAP-Gly core with its N- terminal basic patch) and p150glued (25-144)-microtubule (CAP-Gly core with its C- terminal basic patch) complex were determined at 9.7 Å and 10.2 Å resolution, respectively. These structures revealed that the CAP-Gly domain binds to the flexible C-terminus of the tubulin (known as E-hook) instead of the core of microtubules. Comparison of the p150glued (1-105)-microtubule reconstruction and p150glued (25- 144)-microtubule reconstruction revealed that CAP-Gly interacts with microtubules very flexibly. In addition, the basic extensions of CAP-Gly core was found to induce microtubule lateral association by neutralization of the negatively charged tubulin C- terminus, which acts as an electrostatic shield to avoid the interaction between individual microtubules. Interestingly, p150glued CAP-Gly together with the basic extensions could induce longitudinal interaction of tubulin for forming curved tubulin oligomers at low temperature, and this process happens in a GTP independent manner. Taken together, p150glued CAP-Gly plus its adjacent basic patches interact with the acidic C-terminus of tubulin and promote tubulin polymerization in two directions, by inducing tubulin longitudinal association at low temperature and lateral interaction once temperature change to physiological condition. Our study about p150glued explained how +TIPs regulate microtubule dynamics from a structure point of view. Cilia are rod-like microtubule based structures protruding from most eukaryotic cells. Cilia are assembled and maintained through a bidirectional transport system called intraflagellar transport (IFT) mediated by IFT complexes and molecular motors moving along axonemal microtubules. The IFT complex is composed of at least 22 polypeptides organized into two complexes named IFT-A and IFT-B. The IFT-B complex is further divided into IFT-B1 and IFT-B2. IFT172, one of the IFT-B2 subunits, is the IFT protein with the highest molecular weight. Chlamydomonas IFT172 is a 1755-amino-acid protein that is encoded by FLA11 gene. The N-terminus of IFT172 contains a WD40 domain, which folds into β-propellers structure while its C-terminus shows tetratricopeptide repeats (TPRs) predicted to form α-helical secondary structure. The domain architecture of IFT172 is highly similar to vesicles coat proteins like COPI and clathrin-adaptor subunits. To characterize IFT172, Chlamydomonas IFT172 was expressed from insect cell and further purified. Surprisingly, IFT172 showed lipid association during the purification and the purification products showed round oligomers containing both IFT172 and membrane. To obtain IFT172 in monomer form instead of the oligomers with lipid, n- Dodecyl β-D-maltoside (DDM) was used and by negative-stain electron microscope observation, the IFT172 monomer was found to adopt two conformations: a globular conformation and a rod-shape conformation. Furthermore, giant unilamellar vesicle (GUV) binding assay was employed to assess the interaction of membrane with IFT172. IFT172 showed high membrane binding affinity and clusters on the membrane surface. To investigate the effect of IFT172 on membrane surface closely, IFT172 with Folch fraction I was investigated under the electron microscope. Vesiculation of 18 nm-diameter small vesicles from the large unilamellar vesicle membrane surface was observed. Further studies revealed that the membrane binding property of IFT172 is mediated by its N-terminal β-propellers, but not C-terminal TPRs. Moreover, I demonstrated that IFT57, the direct binding partner of IFT172 within IFT proteins, competes with membrane for IFT172 binding. These results provided the first evidence that IFT172 binds to membrane through its N-terminal WD40 domains directly and it remodels membrane surface in vitro. Investigation of the functions of IFT172 in vivo is needed to address these issues in the future.