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

Abstract

Dsl1p is required for Golgi-endoplasmic reticulum (ER) retrograde transport in yeast. It interacts with the ER resident protein Tip20p and with ␦-COP, a subunit of coatomer, the coat complex of COPI vesicles. To test the significance of these interactions, we mapped the different binding sites and created mutant versions of Dsl1p and ␦-COP, which are unable to bind directly to each other. Three domains were identified in Dsl1p: a Tip20p binding region within the N-terminal 200 residues, a highly acidic region in the center of Dsl1p containing crucial tryptophan residues that is required for binding to ␦-COP and essential for viability, and an evolutionarily well conserved domain at the C terminus. Most importantly, Dsl1p uses the same central acidic domain to interact not only with ␦-COP but also with ␣-COP. Strong interaction with ␣-COP requires the presence of comparable amounts of ⑀-COP or ␤-COP. Thus, the binding characteristics of Dsl1p resemble those of many accessory factors of the clathrin coat. They interact with different layers of the vesicle coat by using tandemly arranged sequence motifs, some of which have dual specificity.The structural integrity of membrane-bound organelles requires the recycling of lipids and proteins. Between Golgi and the endoplasmic reticulum (ER) 1 this retrograde transport is mediated by COPI-coated vesicles (1). The COPI coat from yeast and mammals consists of seven COP proteins (␣-, ␤Ј-, ␤-, ␥-, ␦-, ⑀-, and -COP ϭ coatomer) and the small GTPase ARF1 (2, 3). Sorting of cargo proteins to COPI vesicles can be achieved by direct binding of cargo molecules to coatomer (4 -8). This binding is often mediated by short sequence motifs displayed by cargo molecules (6, 9). The efficient sorting of cargo into COPI vesicles depends on GTP hydrolysis by ARF1 facilitated by a specific GTPase-activating proteins (10).After the uncoating, vesicles are ready to fuse with their specific target membrane (11). Fusion events rely on specific attachment reactions to guarantee that only appropriate membranes can mix. The membrane attachment itself comprises two steps, tethering and docking (12). Both steps involve different sets of proteins. Tethering factors are peripherally membrane-associated protein complexes consisting of up to 10 different subunits, which share little sequence similarity. So far, seven different tethering complexes required for at least five different transport steps were characterized in yeast (13)(14)(15)(16)(17)(18)(19)(20). In some cases, mammalian counterparts of yeast tethering factors were identified (reviewed in Ref. 21).The subsequent docking stage involves specific sets of membrane-anchored proteins, the so-called SNARE proteins (22,23). 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 SNARE or SNARE-like proteins involved in fusion at ...

RINT-1 Regulates the Localization and Entry of ZW10 to the Syntaxin 18 Complex

“…Given that Tip20p directly binds an ER SNARE, Sec20p (Sweet and Pelham, 1993), and that Dsl1p interacts with subunits of coatomer (Andag et al, 2001;Reilly et al, 2001;Andag and Schmitt, 2003), which coats vesicles for retrograde transport (Bonifacino and Glick, 2004;Lee et al, 2004), it has been postulated that the Dsl1p/Tip20p complex functions as a tether in analogy to known tethering complexes Andag and Schmitt, 2003;Lupashin and Sztul, 2005). However, a recent study showed that Tip20p also participates in anterograde transport by prohibiting back-fusion of COPII vesicles that mediate export from the ER (Kamena and Spang, 2004).…”

“…BNIP1 likely corresponds to yeast Sec20p, although their sequences as well as molecular weights are markedly different with each other (Sweet and Pelham, 1992;Nakajima et al, 2004). ZW10 and RINT-1, which were originally characterized as checkpoint proteins (Williams et al, 1992;Chan et al, 2000;Xiao et al, 2001), appear to be equivalent to Dsl1p and Tip20p, respectively (VanRheenen et al, 2001;Andag and Schmitt, 2003;Hirose et al, 2004). The equivalents of all proteins identified in the syntaxin 18 complex are present in the Ufe1p complex (Lewis et al, 1997;Andag et al, 2001;Reilly et al, 2001;Hirose et al, 2004;Nakajima et al, 2004), implying that the ER membrane fusion machinery is conserved during evolution (Kraynack et al, 2005).…”

Coats, Tethers, Rabs, and SNAREs Work Together to Mediate the Intracellular Destination of a Transport Vesicle

“…The d-subunit is a constituent of the F subcomplex of COPI that shares sequence similarities with clathrin adaptor components. The a-COPI subunit belongs to the B subcomplex of the COPI coat, which is thought to function like clathrin (Andag and Schmitt, 2003). Thus, Dsl1p interacts with both the F and B subcomplexes of the COPI vesicle coat.…”

Formation and Maintenance of the Golgi Apparatus in Plant Cells

“…For the Golgi-to-ER COPI vesicles, tethering to the ER membrane is mediated by Dsl1 trimeric complex in yeast cells (Ren et al, 2009). Dsl1 complex is composed of Dsl1, Tip20, and Dsl3/Sec39, and Dsl1 binds to a-and d-COP (Andag and Schmitt, 2003;Reilly et al, 2001). Dsl1 complex stably localizes to the ER membrane independently to any RAB GTPases (Sztul and Lupashin, 2009).…”