A Biochemical Characterization of the Dsl1/αεCOP Interaction

Abstract

The vesicle transport system of eukaryotic cells is essential in mediating the intracellular movement of cargo among membrane-bound organelles. Broadly speaking, vesicle transport involves four steps: budding, transport, tethering, and fusion. Proper orchestration of these steps is critical in maintaining the distinct biochemical composition of organelles. Tethering factors have been implicated in several steps of efficient trafficking including the capture of approaching vesicles, vesicle uncoating, and SNARE protein assembly for fusion. Dsl1, architecturally the simplest of the multi-subunit tethering complexes (MTCs), captures COPIcoated vesicles that are involved in retrograde transport from the Golgi to the ER. Dsl1 forms a tower-like shape: the Tip20 and Sec39 subunits interact with ER SNARE proteins at its base and the Dsl1 subunit binds to COPI vesicles at its tip. Within the Dsl1 subunit, a flexible, negatively charged region, termed the lasso, contains tryptophan residues that interact with the COPI coat. In particular, the W425 residue has been shown to be essential for maintaining interaction with the αεCOP complex. To further understand how Dsl1 binds the COPI coat, gel filtration assays were conducted to determine the effects of altering the charge of the lasso at highly conserved, negatively charged residues. Although single mutations at these residues do not compromise interaction, mutations at multiple residues disrupt Dsl1/αεCOP interaction, suggesting significant changes to the charge of the Dsl1 lasso hinder its function.