The Coatomer-interacting Protein Dsl1p Is Required for Golgi-to-Endoplasmic Reticulum Retrieval in Yeast

Abstract

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a Cterminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.Membrane-bound compartments in eukaryotic cells can fuse directly as shown for the endoplasmic reticulum (ER) 1 and mitotic Golgi fragments as well as endosomal and lysosomal compartments (homotypic fusion; see Ref. 1). However, vectorial transport between distinct compartments mainly involves small coated vesicles whose formation from the donor membrane is mediated by proteinaceous coats, either COPI, COPII, or clathrin. After uncoating, vesicles fuse selectively with an acceptor membrane (heterotypic fusion; see Ref.2). Both homotypic and heterotypic fusion events rely on specific attachment reactions to guarantee that only appropriate membranes can mix. The membrane attachment itself consists of two steps, tethering and docking, involving different sets of proteins (3, 4).Tethering factors are peripherally membrane-associated protein complexes consisting of up to 10 different subunits, which share little sequence similarity.The subsequent docking stage involves specific sets of membrane-anchored proteins, so-called SNARE proteins (SNARE is soluble NSF (for N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) (5-7). SNAREs are inserted into the membrane either by a C-terminal transmembrane domain or through lipid moieties attached to C-terminal cysteine residues. In contrast to the tethering factors, all known SNARE proteins are members of either of three protein families: the syntaxins, the synaptobrevins or VAMPs, and the SNAP-25 family members. To induce membrane fusion, SNARE proteins from apposed membranes must interact in trans. The formation of a stable four-helix bundle may generate enough energy to promote mixing of the lipid bilayer (8 -10).Lipid mixing experiments using SNARE complexes reconstituted into lipid bilayer vesicl...

Dsl1p, an Essential Component of the Golgi-Endoplasmic Reticulum Retrieval System in Yeast, Uses the Same Sequence Motif to Interact with Different Subunits of the COPI Vesicle Coat

“…It is tempting to speculate that this domain in Dsl1p from yeast may also function in the recruitment of cytoskeletal elements. This region has an important function because the previously characterized dsl1-22 mutation is a nonsense mutation that removes the last 30 residues from Dsl1p (32). The alignments of fungal Dsl1 proteins and ZW10 from higher eukaryotes showed that the low complexity region mediating coatomer binding is unique to fungi.…”

“…The apparent lack of function of the double mutation was confirmed independently; ⌬dsl1 spores (dsl1::kanMX) expressing this mutant allele from a centromeric vector could not form colonies. The same plasmid was also unable to suppress the Ts Ϫ -phenotype of dsl1-22 mutant cells (data not shown) (32).…”

“…Expression of either His 6 -tagged or GST fusion proteins was performed using protease-deficient strains BL21DE3 or C41DE3 as described previously (32). Saccharomyces cerevisiae strains used are listed in Table I.…”

“…In contrast, the dsl1-22 mutation showed synthetic interaction with both the ret2-1 and the sec21-1 mutation (32) (33) have isolated fragments of the ␦-COP-encoding RET2 gene in a yeast two-hybrid screen for Dsl1p interacting proteins. The results of this screen indicated that the central part of the protein (residues 156 -398 of 546) contains the Dsl1p binding site.…”

“…Recently, we and others identified a new coatomer-interacting factor, Dsl1p, which is essential for retrieval of proteins from Golgi to ER (31)(32)(33). Dsl1p also interacts with Tip20p (33,34).…”