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COPI Mediates Recycling of an Exocytic SNARE from Endosomes By bioRxiv preprint doi: https://doi.org/10.1101/133546; this version posted May 3, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 1 COPI mediates recycling of an exocytic SNARE from endosomes by 2 recognition of a ubiquitin sorting signal 3 4 Peng Xu1, Hannah M. Hankins1, Chris Macdonald2, Samuel J. Erlinger1, Meredith N. Frazier1, 5 Nicholas S. Diab1, Robert C. Piper2, Lauren P. Jackson1, Jason A. MacGurn3 and Todd R. 6 Graham1* 7 1Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA 8 2Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, 9 USA 10 3Department of Cell and Developmental Biology, Vanderbilt University Medical Center, 11 Nashville, TN 37235, USA 12 *Correspondence to: email: [email protected] 13 14 ABSTRACT 15 The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role 16 in endocytic trafficking. Here we show that COPI mediates delivery of a budding yeast SNARE 17 (Snc1) from early endosomes to the Golgi complex through recognition of a polyubiquitin sorting 18 signal. Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and 19 then recycles through early endosomes back to the Golgi for reuse. Removal of ubiquitin from 20 Snc1, or deletion of a '-COP subunit propeller domain that binds K63-linked polyubiquitin, 21 causes aberrant accumulation of Snc1 in early endosomes. Moreover, replacement of the '-COP 22 propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results 23 indicate that ubiquitination, a modification well known to target membrane proteins to the 24 lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into 25 COPI vesicles at early endosomes for Golgi delivery. 26 27 28 29 30 31 32 1 bioRxiv preprint doi: https://doi.org/10.1101/133546; this version posted May 3, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 33 INTRODUCTION 34 Sequential rounds of vesicle budding and fusion reactions drive protein transport through 35 the secretory and endocytic pathways (Rothman, 1994). Vesicle budding often requires cytosolic 36 coat proteins, such as COPI, COPII or clathrin, that assemble onto the donor organelle to mold the 37 membrane into a tightly curved structure while collecting cargo for inclusion into the nascent 38 vesicle (Faini et al., 2013). Efficient departure of cargo from the donor organelle requires a sorting 39 signal within the protein that is recognized by the coat complexes. For example, the heptameric 40 COPI coat complex assembles onto Golgi membranes where it selects cargo bearing a C-terminal 41 di-lysine (KKxx-COO- or KxKxx-COO-) sorting signal exposed on the cytosolic side of the 42 membrane (Eugster et al., 2004; Letourneur et al., 1994; Waters et al., 1991). The two large COPI 43 subunits, - and ’-COP, each contain a pair of WD40 repeat domains that form twin -propellers 44 used to select cargo by binding to the sorting signal (Jackson, 2014; Jackson et al., 2012). After 45 budding, COPI vesicles uncoat and deliver the di-lysine bearing cargo to the ER by fusing to this 46 acceptor membrane in a SNARE-dependent reaction (Rein et al., 2002; Sudhof and Rothman, 47 2009). 48 Intrinsic to the SNARE hypothesis is the privileged selection of v-SNAREs (also called R- 49 SNAREs) by the vesicle budding machinery to ensure the nascent vesicle can fuse to its target 50 membrane bearing complementary t-SNAREs (Q-SNAREs), and the subsequent need for v- 51 SNARE recycling back to the donor compartment (Miller et al., 2011). v-SNAREs are small, 52 single-pass membrane proteins with the C-terminus embedded within the lumenal space (Weber 53 et al., 1998). Thus, COPI cannot select v-SNAREs using the canonical C-terminal motifs and there 54 is no known sorting signal within a v-SNARE that is recognized by COPI. A further complication 55 with the vesicular transport process is that it leads to depletion of v-SNAREs from the donor 56 membrane and their deposition in the acceptor membrane. Thus, it is essential to recycle the v- 57 SNAREs back to the donor compartment in order to sustain the vesicular transport pathway. 58 Exocytic v-SNAREs that bud from the trans-Golgi network and target vesicles to the plasma 59 membrane have served as models to understand the mechanisms of SNARE recycling, although 60 these studies have primarily focused on how the v-SNAREs are endocytosed from the plasma 61 membrane for delivery to early endosomes (Burston et al., 2009; Lewis et al., 2000; Miller et al., 62 2011). The subsequent step of transport from endosomes back to the Golgi, however, is poorly 63 understood. 2 bioRxiv preprint doi: https://doi.org/10.1101/133546; this version posted May 3, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 64 In the case of the yeast exocytic v-SNARE Snc1, recycling from endosomes back to the 65 Golgi is independent of retromer and clathrin adaptors known to mediate transport of other cargos 66 in these pathways (Lewis et al., 2000). Instead, an F-box protein (Rcy1) (Galan et al., 2001), a 67 phosphatidylserine flippase (Drs2) (Hua et al., 2002; Xu et al., 2013), an ArfGAP (Gcs1) 68 (Robinson et al., 2006) and a sorting nexin complex (Snx4/41) (Lewis et al., 2000; Ma et al., 2017) 69 are required for endosome to Golgi transport of Snc1, although the precise functions for these 70 proteins remain unclear. F-box proteins are best known as substrate-selecting adaptors in Skp1- 71 Cullin-F-box (SCF) E3 ubiquitin (Ub) ligases, but the Rcy1-Skp1 complex plays a role in Snc1 72 recycling that is independent of the cullin subunit or the Cdc34 E2 Ub ligase (Galan et al., 2001). 73 Moreover, ubiquitination of membrane proteins in the endocytic pathway is thought to set a course 74 for their degradation in the lysosome or vacuole via the ESCRT/MVB pathway (MacGurn et al., 75 2012). Thus, it seemed unlikely that Rcy1 mediates ubiquitination of Snc1 in order to recycle this 76 SNARE protein out of the endocytic pathway. Nonetheless, several high-throughput studies have 77 shown that Snc1 is ubiquitinated (Peng et al., 2003; Silva et al., 2015; Swaney et al., 2013), and 78 altering a targeted lysine to arginine (Snc1-K63R) surprisingly perturbed its early endosome (EE) 79 to TGN transport (Chen et al., 2011), suggesting ubiquitin (Ub) conjugation could play a role in 80 Snc1 recycling. 81 Here we show that K63-linked polyubiquitin (polyUb) chains are indeed a sorting signal 82 that drives EE to TGN transport of Snc1, and surprisingly find that COPI mediates this sorting 83 event by direct binding to the polyUb chain. COPI was observed to localize to EEs in mammalian 84 cells more than two decade ago (Aniento et al., 1996; Whitney et al., 1995), but had not previously 85 been shown to localize to EEs in yeast. In addition, COPI was initially thought to mediate transport 86 of proteins from EE to late endosomes in animal cells (Aniento et al., 1996). However, as models 87 for early to late endosome maturation emerged (Scott et al., 2014; Zerial and McBride, 2001), this 88 proposed role for endosomal COPI was abandoned and there remains no clearly defined role for 89 COPI in protein trafficking through the endosomal system. A major impediment to deciphering a 90 function for endosomal COPI is that mutations or knockdown approaches that inactivate COPI 91 grossly disrupt Golgi function. Thus, any endosomal defects observed in COPI-deficient cells 92 could be attributed to an indirect downstream effect of perturbing the Golgi complex. To 93 demonstrate a direct functional role of COPI at endosomes would require specific mutations or 94 variants of COPI that disrupt trafficking through the endosomes while maintaining its function at 3 bioRxiv preprint doi: https://doi.org/10.1101/133546; this version posted May 3, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-ND 4.0 International license. 95 the Golgi. We describe here a set of COPI separation-of-function mutations and fusion proteins 96 that allow us to demonstrate a direct role of this coat at EEs to select cargo bearing K63-linked Ub 97 chains. 98 99 100 101 RESULTS 102 103 Snc1 ubiquitination is required for EE to TGN transport. 104 Snc1 is an exocytic v-SNARE that is incorporated into vesicles budding from the trans- 105 Golgi network (TGN) to drive fusion of these vesicles with the plasma membrane (Gerst et al., 106 1992). Afterwards, Snc1 is endocytosed, delivered to an early endosome population marked by the 107 t-SNARE Tlg1 and then delivered back to the TGN for reuse in exocytosis (Lewis et al., 2000; 108 Robinson et al., 2006). In wild-type (WT) cells, nearly half of GFP-Snc1 localizes to the plasma 109 membrane (preferentially at the buds relative to the mother cell) and the remainder is observed in 110 early endosomes and Golgi cisternae, which appear as small punctae (Figure 1A, B).
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