| This dissertation is a study of the Golgi-associated retrograde protein (GARP) complex, a heterotetrameric membrane tethering complex that functions in traffic from endosomes to the trans-Golgi network. While subunits from other tethering complexes, have been structurally characterized, there have been no structural data regarding GARP subunits. I performed extensive expression and purification studies on all GARP subunits from both yeast and humans and purified successfully soluble Vps53 and Vps52, which were used in biochemistry experiments to investigate small GTPase binding. I also purified larger ternary and quaternary subcomplexes.;I used the soluble subunits as the basis for crystallographic studies and here I present the structure of a C-terminal fragment of the Vps53 subunit, important for binding endosome-derived vesicles, at a resolution of 2.9 A. I show that the C-terminus consists of two alpha-helical bundles arranged in tandem, and I identify a highly conserved surface patch, which may play a role in vesicle recognition. Mutations of the surface result in defects in membrane traffic, as shown by the carboxypeptidase Y assay. The fold of the Vps53 C-terminus is strongly reminiscent of proteins that belong to three other tethering complexes---Dsl1, COG, and the exocyst---thought to share a common evolutionary origin. Thus, the structure of the Vps53 C-terminus suggests that GARP belongs to this family of complexes.;I used the ternary and quaternary complexes as the basis for electron microscopy studies and here I present preliminary data on these GARP complexes. I show single particles of GARP that resemble a Y, similar to what has been observed with a COG subcomplex. Together these structural studies show that at the tertiary level, GARP is similar to this larger family of complexes, and suggest that at the quaternary level, GARP has similarities with at least one of these complexes. Clarifying these similarities and differences will help elucidate the commonalities across all tethering complexes, as well as nuances specific to tethering in the retrograde transport pathway.;Finally, I show preliminary biochemical data of exocyst subcomplexes, towards reconstituting the entire exocyst. I purified soluble Sec10/Sec15, and in collaboration, soluble Sec6/Sec8, and Sec5/Sec6/Sec8 subcomplexes. Using these and other reported interactions using recombinant proteins, I present a refined interaction map for exocyst subunits.;In total, this work provides a starting point for future biochemical, structural, and functional studies aimed at understanding tethering complexes. |
