The Dsl1 Tethering Complex Actively Participates in Soluble NSF (N-Ethylmaleimide-sensitive Factor) Attachment Protein Receptor (SNARE) Complex Assembly at the Endoplasmic Reticulum in Saccharomyces cerevisiae

Melanie Diefenbacher,Holmfridur Thorsteinsdottir,Anne Spang

doi:10.1074/jbc.m110.215657

Abstract

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 Golgi-derived 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 temperature-sensitive 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, particularly 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 the coatomer coat complex, 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.

Highlights

University of Basel. □S The on-line version of this article contains supplemental Tables S1–S3 and Figs
We investigated the function of the Dsl1 complex member Tip20p by analyzing the phenotypes of tip20 mutants and found that they interfered with the proper assembly of transSNARE complexes at the ER (Fig. 5F)
Trans-SNARE complexes consisting of the t-SNAREs Sec20p, Ufe1p, and Use1p and the vesicle SNARE (v-SNARE) Sec22p and/or Bet1p promote fusion of Golgi-derived COPI-coated vesicles with the ER (24, 30 –32)

Summary

Introduction

University of Basel. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S3 and Figs. The SNARE complexes involved in the fusion of Golgi-derived COPI vesicles with the ER are the v-SNARE Sec22p and the three t-SNAREs Sec20p, Ufe1p, and Use1p (24, 30 –32) Another v-SNARE, Bet1p, could participate in the fusion of retrograde transport carriers with the ER [33]. The trans-SNARE complexes formed during the fusion of COPII vesicles at the Golgi contain the t-SNARE Sed5p and the v-SNAREs Bos1p, Bet1p, and Sec22p or Ykt6p, which seem to be functionally redundant in this process in vivo [18, 24]; in this case, the v-SNAREs seem to provide three helices and the t-SNARE only one. The Dsl complex appears to have two functions: one is tethering COPI vesicles through Dsl1p and the second is increasing the efficiency of the fusion process through acceleration of SNARE complex assembly

Objectives

Results

Conclusion