Structural studies of the endoplasmic reticulum to Golgi vesicular transport proteins Sed5p and Cog2p of Saccharomyces cerevisiae

Eukaryotic cells distribute proteins and lipids among intracellular compartments by means of lipid vesicles that bud from one compartment and fuse with another. A fundamental question in cell biology is how these vesicles accomplish the proper sorting of cargo among the many compartments within a cell. The initial recognition between cargo vesicles and their intracellular targets is thought to be mediated by a conserved oligomeric Golgi (COG) complex. To begin to understand the specificity and mechanism of vesicle targeting, we have initiated studies of a component of this complex. Cog2p is an essential 32 kD protein required for vesicle-target binding in in vitro assays. It interacts with a number of other proteins important for vesicle docking and fusion. To develop a molecular framework for understanding Cog2p function, we aim to determine its three-dimensional structure. Cog2 was expressed and purified to homogeneity from E. coli. The full-length protein forms oligomers that vary based on solution conditions. The folded core of the protein was determined by limited proteolysis, resulting in two related fragments containing the C-terminus of Cog2p. Both yielded crystals that, unfortunately, do not diffract x-rays beyond 8 Å resolution. One fragment, comprising residues 74–275, appears to contain all of the folded α-helical structure present in the full-length protein, as judged by circular dichroism spectroscopy. Cog2p_74–275 can rescue cog2Δ strains from lethality and is monomeric at millimolar concentrations, as assayed by dynamic light scattering. At 23.4 kDa, it is a reasonable candidate for solution structure determination, which is currently underway.; Cog2p has genetic and physical interactions with the Golgi docking protein Sed5p. Sed5p was expressed and purified to homogeneity from E. coli . The cytoplasmic domain of Sed5p is prone to aggregation and degradation. Limited proteolysis allowed the identification of the folded core of Sed5p, which resides in the N-terminal regulatory domain of the protein. Crystallization trials on Sed5p have not been successful. We have demonstrated that Sed5p and Cog2p interact directly using GST-pulldown assays. With the structure and binding studies of Cog2p with other proteins required for vesicular transport, we will be able to explore COG function in detail.