Dsl1p, Tip20p, and the Novel Dsl3(Sec39) Protein Are Required for the Stability of the Q/t-SNARE Complex at the Endoplasmic Reticulum in Yeast

Abstract

The "Dsl1p complex" in Saccharomyces cerevisiae, consisting of Dsl1p and Tip20p, is involved in Golgi-ER retrograde transport and it is functionally conserved from yeast to mammalian cells. To further characterize this complex, we analyzed the function of Dsl3p, a protein that interacts with Dsl1p in yeast two hybrids screens. DSL3, recently identified in a genome wide analysis of essential genes as SEC39, encodes a cytosolic protein of 82 kDa that is peripherally associated with membranes derived from the ER. There is strong genetic interaction between DSL3 and other factors required for Golgi-ER retrograde transport. Size exclusion chromatography and affinity purification approaches confirmed that Dsl3p is associated with subunits of the "Dsl1p complex." The complex also includes the Q/t-SNARE proteins, Use1p, Sec20p, and Ufe1p, integral membrane proteins that constitute the trimeric acceptor for R/v-SNAREs on Golgi-derived vesicles at the ER. Using mutants, we performed a detailed analysis of interactions between subunits of the Dsl1p complex and the ER-localized SNARE proteins. This analysis showed that both Dsl1p and Dsl3p are required for the stable interaction of the SNARE Use1p with a central subcomplex consisting of Tip20p and the SNARE proteins Ufe1p and Sec20p. INTRODUCTIONThe protein trafficking pathway in the yeast S. cerevisiae is composed of several distinct membrane-bounded compartments, which interact via a bidirectional flow of membranebounded vesicles in a process referred to as vesicular transport (Kaiser and Schekman, 1990;Rothman and Orci, 1992;Ferro-Novick and Jahn, 1994;Rothman, 1994;Waters and Hughson, 2000). Briefly, secretory proteins destined for transport must be sorted away from resident proteins, packaged into the proper cargo vesicles, and subsequently, delivered to the correct target membrane (Palade, 1975). Each step of this process must be tightly regulated to ensure efficient secretion and maintenance of the distinct cellular compartments. Transport in the retrograde direction ensures further rounds of anterograde transport by recycling components of the transport machinery, recovering wayward proteins and maintaining the balance of lipids between the distinct compartments of the pathway.The cell makes use of a variety of coated vesicles to transport proteins, whose formation is nucleated by the action of small GTP-binding proteins. Specifically, vesicle budding in the retrograde direction from the Golgi to the ER involves a heptameric coat protein complex called COPI (Waters et al., 1991;Stenbeck et al., 1993;Letourneur et al., 1994;Barlowe, 2000). The COPI coat consists of coatomer, an ϳ700 -800 kDa protein complex comprised of an equimolar assembly of ␣-, ␤-, ␤'-, ␥-, ␦-, ⑀-, and -COP (Cop1[Ret1]p, Sec26p, Sec27p, Sec21p, Ret2p, Sec28p, and Ret3p, respectively, in yeast) and a small ras-like GTPase termed Arf (encoded by ARF1 or ARF2).Current models of vesicular transport propose that the vesicle coat is removed soon after vesicles are formed (although this has not been di...

The COG complex interacts with multiple Golgi SNAREs and enhances fusogenic SNARE complexes assembly

“…The Tip20 and Sec39 subunits of the Dsl1 complex, which regulates Golgi-to-ER transport and accelerates SNARE complex assembly in vitro, interact directly with the N-terminal regulatory domains of the tSNAREs Use1 and Sec20. These interactions are necessary for SNARE complex assembly (Diefenbacher et al, 2011;Kraynack et al, 2005;Ren et al, 2009;Tripathi et al, 2009). Similarly, all four subunits of mammalian GARP interact directly with Stx6, Stx16 and VAMP4, and regulate Stx6 SNARE complex assembly (Pérez-Victoria and Bonifacino, 2009;Pérez-Victoria et al, 2010).…”

Secretory Protein Biogenesis and Traffic in the Early Secretory Pathway

“…Dsl1 forms a complex with Dsl3/Sec39 and Tip20 to form the Dsl1 complex, another member of the CATCHR family of tethering complexes noted for their extended helical rod structures (Lees et al 2010). Further genetic and biochemical dissection of these proteins converged on a role in retrograde transport from the Golgi to the ER: tip20 and dsl1 mutants showed genetic interactions with a variety of ER-Golgi SNAREs (Sweet and Pelham 1993;Andag et al 2001;Kraynack et al 2005), tip20 mutants showed defects in fusion of COPI vesicles (Kamena and Spang 2004), the Dsl1 complex was localized to the ER (Kraynack et al 2005), and Dsl1 interacts directly with multiple components of the COPI coat (Andag and Schmitt 2003).…”

“…Sec39 itself binds to the N-terminal domain of the ER resident SNARE, Use1, via a region that likely lies proximal to the membrane , and Tip20 contains a second SNARE-binding site, interacting with the N-terminal domain of Sec20 ). In addition to binding individual SNAREs, the Dsl1 complex also promotes SNARE assembly and thus may serve two roles in fusion: maintaining individual SNAREs in an unpaired, receptive state, and scaffolding assembly of the fusogenic SNARE complex to promote fusion (Kraynack et al 2005;Ren et al 2009). An additional role in vesicle uncoating is suggested by the tendency of vesicles to accumulate en masse under conditions of Dsl1 depletion (Zink et al 2009): COPI shedding might be assisted by a Dsl1-COPI interaction that would prevent repolymerization of disassembled coat subunits, or could be driven by conformational changes in the Dsl1 complex that would capitalize on the ability of Dsl1 to interact with both the outer a-/e-COPI domain and a second site on the inner d-COP subunit to prize the coat from the membrane Zink et al 2009).…”

The Dsl1 Protein Tethering Complex Is a Resident Endoplasmic Reticulum Complex, Which Interacts with Five Soluble NSF (N-Ethylmaleimide-sensitive Factor) Attachment Protein Receptors (SNAREs)

“…Moreover, the interaction of Dsl1 and COPI suggested that Dsl1 could be recruited to retrograde vesicles. We therefore took advantage of a COPII budding assay (22) to monitor the incorporation of selected subunits of the Dsl1 complex and SNAREs (Fig. 4A).…”