Uncoating Vesicle Transport: Protemic Analysis of Potential Dsl1 Tethering Interactions

Abstract

Eukaryotic cells use vesicle-mediated transport to traffic membrane and cargo materials between intracellular membrane-bound compartments. Tethering factors are thought to participate in several steps required for efficient trafficking by forming initial contacts with vesicles, facilitating removal of vesicle coats, and organizing assembly of SNAREs, the proteins that drive membrane fusion. Dsll is the tethering factor essential to retrograde traffic from the Golgi to the ER. It is the architecturally simplest multisubunit tethering complex (MTC), comprising three essential proteins Dsll, Tip20, and Sec39. The Dsll complex is a tower-like structure that binds to ER SNAREs via Tip20 and Sec39. At the membrane-distal tip of the complex, Dsll is thought to interact with incoming vesicles by binding to the COPI vesicle coat via acidic tryptophan motifs contained an unstructured loop. Surprisingly, the lasso is not essential to Dsll function, but is made essential by the deletion of the C-termainal E domain. This implies that the lasso and E domain have related functions that are each sufficient to maintain viability in the absence of the other. We have undertaken a proteomic approach to investigate the potential function of these two regions of Dsll by comparing protein binding interactions associated with wild-type Dsll and the deletion mutant to identify interactions that are specific to the lasso orE domain. We have demonstrated a lasso-specific interaction with coatomer. Mutagenesis experiments corroborate these findings by demonstrating a critical role for a-COP interaction with the lasso. Though protein binding partners of E domain were not identified, it is clear that it does not bind coatomer in a manner similar to the lasso.