THE DSL1 TETHERING COMPLEX ACTIVELY PARTICIPATES IN SNARE COMPLEX ASSEMBLY AT THE ENDOPLASMIC RETICULUM IN S. CEREVISIAE

Intracellular transport is largely dependent on vesicles that bud off from one compartment and fuse with the target compartment. The first contact of an incoming vesicle with the target membrane is mediated by tethering factors. The tethering factor responsible for recruiting Golgiderived vesicles to the ER is the Dsl1 tethering complex, which is comprised of the essential proteins Dsl1p, Dsl3p and Tip20p. We investigated the role of the Tip20p subunit at the ER by analyzing two mutants, tip20-5 and tip20-8. Both mutants contained multiple mutations that were scattered throughout the TIP20 sequence. Individual mutations could not reproduce the ts-phenotype of tip20-5 and tip20-8, indicating that the overall structure of Tip20p might be altered in the mutants. Using molecular dynamics simulations comparing Tip20p and Tip20-8p revealed that some regions, in particular the N-terminal domain and parts of the stalk region were more flexible in the mutant protein, consistent with its increased susceptibility to proteolysis. Both Tip20-5p and Tip20-8p mutants prevented proper ER trans-SNARE complex assembly in vitro. Moreover, Tip20p mutant proteins disturbed the interaction between Dsl1p and coatomer, indicating that the Dsl1p-coatomer interaction could be stabilized or regulated by Tip20p. We provide evidence for a direct role of the Dsl1 complex, in particular Tip20p, in the formation and stabilization of ER SNARE complexes. Introduction The correct targeting and delivery of proteins and lipids to various organelles, including the cell membrane, is an essential process in eukaryotic cells. Vectorial transport ensures directionality and provides the order in which proteins travel through organelles along the secretory pathway. Traffic between different membrane-bound compartments is mediated mostly by transport vesicles. The basic principles for generation and consumption of COPII and COPI vesicles that operate in the ER-Golgi shuttle and within the Golgi complex are very similar. In either case, a small GTPase (Sar1p for COPII and Arf1p for COPI) is recruited to the donor membrane and activated. The GTPase in turn recruits cargo, SNAREs -which are important for the subsequent fusion eventand additional coat proteins (1-3). In addition to Sar1p, the COPII coat consists of the Sec23/34 and Sec13/31 subcomplexes, whereas COPI vesicles require Arf1p, an ArfGAP and the heptameric coatomer complex (4-8). The polymerization of coat proteins deforms the membrane, and ultimately, transport vesicles are released. The exact timing for uncoating to vesicles remains elusive, but some partial uncoating may occur already during the budding process and could be completed after tethering to the target membrane (3,9-11). The first contact of the vesicle with the acceptor membrane is mediated by tethering factors bringing the vesicle closer to the membrane at which a vesicle SNARE (v-SNARE) and the SNAREs on the target membrane (t-SNAREs) form a four-helix bundle (trans-SNARE complex) thereby promoting fusion of the lipid bilayers (12-15). Some SNAREs can participate in multiple fusion events at different compartments and those can act redundantly to other SNAREs (16-21). Therefore, additional factors are needed to provide specificity in the fusion process. Rab/Ypt-GTPases, tethering factors as well as Sec1/Munc18 (SM) proteins have been shown to orchestrate, stabilize and proofread the assembly of cognate v-t-SNARE complexes (22-29). http://www.jbc. org/cgi/doi/10.1074/jbc.M110.215657 The latest version is at JBC Papers in Press. Published on April 11, 2011 as Manuscript M110.215657