Some cells release molecules, such as hormones and neurotransmitters, to signal to other cells and influence how they work. As part of the release process, these molecules are packaged into small, balloon-like structures called vesicles. Such vesicles move around within cells and are able to find the right place to release their contents to the outside.

A cellular compartment called the Golgi complex helps to prepare proteins for release from the cell. Vesicles can bind to tethering proteins on the surface of the Golgi, but it was not clear how these proteins are able to capture the correct kind of vesicle. The prediction was that the proteins are rigid, shaped like pipe cleaners that stick out from the Golgi as a meshwork that traps vesicles.

Cheung et al. isolated a specific Golgi tethering protein (called GCC185) from cultured human cells and used a technique called atomic force microscopy to visualize its structure. This revealed that this protein is not rod-like; it is instead rather floppy, and has two arms at one end that may ‘hug’ the incoming vesicle. Cheung et al. showed that this protein needs its middle portion to be floppy to work correctly. This changes the way we think about how vesicles are able to find their corresponding targets on different compartments inside cells.

Further experiments are now needed to answer a number of questions. What does the tether look like when actually bound to a vesicle? What happens after the vesicle binds – how does the tether let go? What other components are needed for vesicle capture and release?

Membrane trafficking involves the collection of cargo into transport vesicles, movement of vesicles along cytoskeletal tracks, and tethering, docking and fusion of vesicles at their target membranes. Tethering is the process by which partner membranes are brought together into close proximity to permit their subsequent fusion (Pfeffer, 1999; Sztul and Lupashin, 2006; Yu and Hughson, 2010). Two classes of transport vesicle tethers have been described to date: multi-subunit complexes such as the Exocyst, COG, Dsl1 and TRAPP complexes (Bröcker et al., 2010), and larger, dimeric coiled-coil containing proteins such as p115 and EEA1 (Sztul and Lupashin, 2006; Munro, 2011). Both classes share the ability to bind to Rab GTPases, SNARE proteins and vesicle coat complexes, suggesting that they play important roles in coordinating molecular events important for target recognition and membrane fusion (Short et al., 2005; Cai et al., 2007; Sinka et al., 2008; Hayes et al., 2009; Hughson and Reinisch, 2010).

Golgins are long, Golgi-associated proteins that contain a high proportion of sequences that are predicted to form coiled coils (Munro, 2011; Short et al., 2005). They are anchored to the Golgi via their C-termini, and models suggest that they protrude long distances, relying on the relative rigidity of the coiled coil structure to provide a meshwork that can capture vesicles in the vicinity of the Golgi (Yu and Hughson, 2010; Munro, 2011). GCC185 is a Golgin required for the transport of mannose 6-phosphate receptors (MPRs) from late endosomes to the trans Golgi network (Reddy, 2006; Derby et al., 2007). Cells depleted of GCC185 accumulate MPRs in Rab9 GTPase- and AP-1-decorated transport carriers (Hayes et al., 2009; Reddy, 2006; Brown et al., 2011), providing an assay for GCC185 tethering capacity. GCC185 is likely anchored on the TGN surface by cooperative binding of its C-terminus to both Rab6 and Arl1 GTPases (Burguete et al., 2008). We (Reddy, 2006; Burguete et al., 2008) and others (Derby, 2004) have shown that GCC185 binds Arl1 rather weakly, but Rab6A binding greatly enhances Arl1 interaction (Burguete et al., 2008). Arl1’s role in anchoring GCC185 was questioned because it seemed not to be required if siRNA-depleted by 80% (Houghton et al., 2009); in fact, 90% depletion of Arl1 is required to displace GCC185 from membranes and to detect its role in Golgi localization of the GCC185 C-terminus (Burguete et al., 2008).

To date, we have little (if any) information, whether Golgins are actually as long as predicted, whether they are relatively rigid, or even where or how they bind transport vesicles. Moreover, once they bind vesicles, what happens next? Many coiled-coil tethers contain putative hinges, located between predicted rod-like, coiled-coil domains (Barr and Short, 2003; Cai et al., 2007; Lupas et al., 1991; Munro, 2011; Pfeffer, 1999). Do these regions function as hinges? We have taken a multidisciplinary approach to investigate the mechanism of vesicle tethering by a trans Golgi network (TGN)-associated Golgin, GCC185. Our data provide novel insight into the tethering process and suggest that the TGN Golgin, GCC185, uses an unexpected mechanism to accomplish vesicle tethering.

Secondary structure prediction algorithms (Lupas et al., 1991) suggest that GCC185 may be almost entirely coiled-coil, perhaps >200 nm in length. To determine the protein’s actual length, we expressed and purified an N-terminally GFP-FLAG-tagged, GCC185 wild type in HEK293 cells. To test models of vesicle tethering that invoke tether bending, we also generated a '△hinge' construct missing two potential hinge domains near the middle of the protein that were selected based upon predicted breaks in coiled coil sequences (Figure 1; △751-805, △890-939). The wild type protein migrated at ~220 kD upon SDS-PAGE; GCC185 △hinge was somewhat smaller (Figure 2A). Purified GCC185 was imaged by atomic force microscopy (AFM) after application onto a mica surface. AFM of GCC185 under physiological buffer conditions revealed the molecule as a parallel dimer of ~145 nm in length with splayed ends (Figure 2B,D; Figure 3, Figure 3—figure supplement 1–3). The N-terminal, globular GFP moiety could often be visualized; the C-terminal, smaller 'GRIP domain' could also be detected. GCC185 contains numerous cysteine residues (Figure 3—figure supplement 1,2); it was important to include dithiothreitol to mimic the reducing environment of the cytoplasm for all experiments.

Figure 1

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Figure 2

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Figure 3 with 4 supplements see all

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Eighty five percent of the molecules showed splaying of at least one end of the dimeric coiled coil, and 38% showed splaying of both ends (Figure 3). It is important to note that the N-terminal GFP used in AFM experiments did not impede the ability of the N-terminus to form a coiled coil because many fewer splays and central bubbles were seen in initial experiments when GCC185 was purified in the absence of dithiothreitol (Figure 3—figure supplement 3). Without DTT, GCC185 appeared rod-like.

Remarkably, half of the GCC185 molecules displayed central unwinding of the coiled coil structure (Figures 2,3). To our knowledge, such unwinding in a coiled coil has not been detected previously. The unwound 'bubble' region was less frequently observed in GCC185 △hinge protein preparations (Figure 2C,D) and thus not a general artifact of protein deposition on mica; the mean size of the bubble was ~27 nm in wild type versus ~17 nm in △hinge molecules, with a much sharper size distribution in the latter case, and only 20% of the △hinge molecules showing any bubble at all (Figure 2C,E). The normalized splay-to-splay distance (Figure 2F) provides information about the molecule’s tendency to bend in this region; rigid (or elongated) molecules have a longer splay-to-splay distance (approaching a value of 1) than bent molecules. △hinge proteins all showed the longest mean, normalized splay-to-splay distances; using this metric, wild type molecules with bubbles were more bent than those without bubbles (Figure 2F). These data show that GCC185 is a flexible molecule and its tendency to bend is contributed by the so-called 'hinge' residues 751-805 and 890-939.

That the molecules detected by AFM represent individual dimers rather than end-to-end tetramers was determined by introduction of an HA epitope tag into the central hinge region (Figure 4A); antibody labeling of such molecules was readily detected by AFM and showed antibodies at the position expected for the epitope tag location within a parallel dimer (Figure 4B-D). These data also confirm the location of the bubble in relation to the position of the epitope tag at residue 806.

Figure 4

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AFM also revealed that purified GFP-FLAG-GCC185 has an overall mean length of ~145 nm (Figure 3—figure supplement 1,2,4). GCC185 fragments were also generated that span the length of the protein (residues 1-358, 394-751, 1-889 and 890-1684). Purified fragments spanning the first and second, predicted coiled-coils were similar in length; the N-terminal half of GCC185 (1-889) was longer than half the length of the complete protein (mean length = 91.2 nm; Figure 3— figure supplement 2, 4). The sum of the lengths of GCC185 fragments could account for the length of the full-length protein determined by AFM. However, full-length GCC185 and all fragments were ~30% shorter than the predicted lengths for coiled-coils with the same number of residues (the cartoons in Figure 3—figure supplement 1,2 are drawn to scale for each portion of the molecules and summarized in Figure 3—figure supplement 4). These data indicate that GCC185’s N-terminus contains more coiled coil than the C-terminal half, and the entire protein contains less total coiled-coil structure than originally predicted.

To investigate the functional importance of the bubble-forming sequences (and their need to flex) for GCC185’s tethering function, we tested the ability of the hinge deletion mutant (Figure 1) to rescue transport of MPRs from late endosomes to the Golgi in GCC185-depleted cells. In these experiments, immunoblots confirmed ~85% depletion of endogenous GCC185 protein (Figure 5B inset).

Figure 5

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As expected, in depleted cells, MPRs were detected in numerous, peripheral transport intermediates; the Golgi ribbon was also dispersed (Figure 5A,B; Hayes et al., 2009; Reddy, 2006; Brown et al., 2011). This phenotype was readily reversed in cells co-expressing an siRNA-resistant Myc-GCC185 protein (Figure 5A,B). In contrast, much less rescue was detected when the △hinge mutant protein, expressed at the same level (Figure 5C), was tested for rescue capacity (Figure 5A,B). Moreover, a protein in which putative hinge sequences were replaced with unstructured gly-gly-ser repeats of the same length (Figure 1) was fully capable of rescuing both transport vesicle receipt and normal Golgi morphology (Figure 5A,B). Quantification verified that all three myc-tagged, rescue constructs were expressed at similar levels in the individual, scored cells (Figure 5C; overall rescue construct transfection efficiency 60%), at no more than 3-5 fold the level of the endogenous protein for the total pool of cells. These experiments demonstrate the importance of hinge sequences for GCC185-mediated vesicle tethering, and show that these sequences provide flexibility rather than a binding site for another cellular component.

The above experiments show that GCC185 tethering requires central flexibility, implying that GCC185 may flex in the middle—to bring N-terminally bound cargo closer to the target membrane (and to the tether’s C-terminus). This was tested using a 'proximity ligation assay' (PLA) to monitor the proximity of GCC185’s N- and C-termini on the surface of the Golgi in cells. Briefly, cells are labeled with primary antibodies from different species that are specific for the two domains to be tested; secondary antibodies are added that have short oligonucleotides attached. A circularizing oligonucleotide is added, ligated, and amplified; the product is detected by hybridization to a fluorescent oligonucleotide. Epitopes that are within 40 nm of each other are detected as a fluorescent spot. The total intensity of PLA signal per cell can then be quantified (Carpenter et al., 2006).

Using mouse anti-GCC185 N-terminus and rabbit anti-GCC185 C-terminus antibodies, PLA signal was readily detected on Golgi complexes of individual cells (Figure 6A). In contrast, no signal was obtained using mouse anti-Golgin 245 C-terminus and rabbit anti-GCC185 C-terminus antibodies, even though both proteins are localized to the trans Golgi network. Similarly, mouse anti-cis-Golgi GM130 antibodies yielded only a very weak PLA signal when used in combination with rabbit anti-GCC185 antibodies. The specificity of the anti-GCC185 antibodies was confirmed using purified GCC185 N- and C-terminal domain fragments (residues 1–358 or 1342–1684; Figure 6E). Quantification of the PLA data confirmed specific PLA labeling for reactions with the GCC185 N- and C-terminal domain antibodies (Figure 6B), consistent with proximity of the N- and C-termini of this protein on the Golgi in cells.

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We also tested the relative ability of △hinge GCC185 to yield a proximity signal for its N- and C-termini (Figure 6C,D). Cells expressing GFP-GCC185 wild type and GFP-GCC185 △hinge constructs showed similar levels of protein expression (Figure 6D, X-axis). However, GFP-GCC185-△hinge yielded a significantly weaker PLA signal when compared with cells expressing GFP-GCC185 (Figure 6C,D). This suggests strongly that the flexibility-conferring regions facilitate the ability of the N- and C-termini to achieve proximity on the Golgi. Quantitation of PLA signal intensity as a function of protein expression confirmed the importance of the hinge region for proximity ligation (Figure 6D). Only at higher expression levels was proximity detected for the GCC185-△hinge construct; at high expression levels, these are likely to represent inter- rather than intra-molecular processes. These experiments show that the presence of a flexible region is needed for GCC185 function and permits the N- and C-termini to become closer together, on the Golgi, in cells. It should be noted that proximity ligation could indicate bending/flexing of individual proteins or inter-digitation of two different proteins in anti-parallel orientation; we favor the former explanation, as it is hard to imagine that hinge deletion would influence the possibility of inter-digitation.

Vesicle capture by GCC185 splayed N-termini

Current models posit that a tethering protein should capture vesicles at one end and be anchored to the target membrane at the other end. For GCC185, the N-terminus would therefore be predicted to bind vesicles. We used differential centrifugation to isolate a crude vesicle fraction carrying the cargoes, GFP-Rab9 (Figure 7B) and endogenous cation-independent MPR (CI-MPR; Figure 7C) and Rab9 (Figure 7D) proteins. We then compared the ability of GCC185 N-terminal residues 1-358 or (similarly sized) internal residues 1032–1331 (Figure 7A) to bind these vesicles after immobilization on glutathione-Sepharose. Figures 7B-D show that vesicles were indeed captured by the N-terminal 1–358 fragment but not by the internal fragment of similar length. Interestingly, the 1–358 sequences tethered most effectively when the obligate GST dimer was located at the fragment’s C-terminus (1–358-GST) rather than the N-terminus (GST-1–358; Figure 7C). Specificity control experiments showed that neither early endosome (EEA1) nor endoplasmic reticulum (calnexin) markers were captured by the constructs tested (Figure 7C). N-terminal sequences are essential for GCC185 function in cells (Hayes et al., 2009), consistent with their ability to bind transport vesicles in vitro.

Figure 7

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The functional significance of N-terminal splaying detected in 80% of purified GCC185 molecules (Figure 2,3) is confirmed by the finding that the fragment capable of splaying at the N-terminus (1–358-GST) showed highest tethering activity (Figure 7C,D). Although we cannot rule out completely, the possibility of GST-steric hindrance for vesicle binding when present at the N-terminus, these data suggest that free N-termini represent both the active and most abundant form of this tethering protein. These experiments show that GCC185 N-terminal sequences can bind cargo—essential for a bona fide tethering protein. Moreover, this represents the first direct demonstration of in vitro vesicle tethering by a TGN localized, GRIP-domain containing tethering protein.

We have shown, for the first time, that flexibility in a tethering protein is required for its functionality in cells, both in supporting the receipt of transport vesicles at the Golgi and maintaining Golgi ribbon structure. We have shown that transport vesicles can bind to GCC185’s N-terminus, and seem to prefer to bind to splayed ends of this dimeric, coiled coil tether. The presence of multiple Rab-binding sites along the length of individual Golgin proteins (Sinka et al., 2008; Hayes et al., 2009; Short et al., 2001) has led others to suggest that vesicles may hop along individual tethers (Barr and Short, 2003) or be captured by Golgin ‘tentacles’ within a potential Golgin meshwork towards the target membrane (Munro, 2011). Such a model considers tethers as rigid, extended rods (Figure 8, left). Our data favor a model whereby GCC185 is C-terminally anchored to the TGN through Rab6 and Arl1 GTPases (Burguete et al., 2008) and captures Rab9-decorated transport vesicles (Hayes et al., 2009; Reddy, 2006) by binding them at the N-terminus and/or near the hinge region (Brown et al., 2011); importantly, the central bubble gives significant flexibility to the protein and would allow it to collapse onto the Golgi surface, bringing vesicles close to the trans Golgi target membrane (Figure 8). GCC185 can bind multiple Rab GTPases along its length (Hayes et al., 2009), as well as syntaxin16, Arl1 and Arl4 GTPases (Burguete et al., 2008; Ganley et al., 2008; Lin et al., 2011). GCC185 also binds to CLASP proteins that catalyze microtubule polymerization from the Golgi surface (Efimov et al., 2007). These additional interactions will all pull the tether towards the Golgi surface, bringing N-terminally bound vesicles closer to the Golgi surface for productive vesicle docking.

Figure 8

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We have shown here that the functionally essential, GCC185 N-terminus has the capacity to bind to transport vesicles in vitro. N-terminal vesicle binding is shared with GMAP-210, a Golgin localized to the cis-Golgi (Sato et al., 2015); for other Golgins, it is presumed due to binding sites for various coat proteins. In previous work (Brown et al., 2011), we showed that GCC185 residues 938–1032 comprise an AP-1 binding site that is also needed for GCC185 vesicle tethering (blue bar in Figure 1A); the transport vesicles are AP-1 decorated, thus this was presumed to represent a possible vesicle binding site. This AP-1 binding site is located immediately downstream of the regions deleted in the △hinge protein studied here (751-805, 890939) (Figure 1A). Moreover, an apparently dispensible, Rab9 GTPase binding site is located directly between the two deletions made to generate the △hinge mutant (orange bar, Figure 1A). Binding of Rab9 to that site could influence the ability of that part of the protein to form a bubble, and could influence the efficiency of tethering on a time scale finer than that assayed here and previously. Both Rab9 and AP-1 are present on the transport vesicles captured, but we do not yet know the precise molecular interaction underlying the ability of the N-terminal domain to bind vesicles. That a splayed N-terminus is utilized suggests that the tether gains efficiency by avidity, and can reach out to 'hug' the vesicle. How the various binding sites along GCC185 coordinate interactions with the vesicle and the target membrane to accomplish vesicle tethering represents an important area for future work.

Given the presence of AP-1 and Rab9 binding sites adjacent to the central bubble (Figure 1A) and the additional capacity of the splayed N-terminus to capture transport vesicles, it is also possible that rather than being held near GCC185’s N-terminus, the vesicle could bind primarily near the bubble while still being enwrapped by the N-terminal arms. The model in Figure 8 is drawn to scale and assumes that the transport vesicles are ~50 nm in diameter (Barbero, 2002); they need not be larger to be able to occupy both potential vesicle binding domains. The AP-1 binding site (939–1,031)is essential for GCC185’s role in vesicle tethering but not Golgi ribbon maintenance (Brown et al., 2011), and must be considered in any model of GCC185 vesicle tethering.

Like GCC185, the other human GRIP domain Golgins are also predicted to contain a high proportion of coiled coil sequences. Golgin-245 shares with GCC185 two very obvious breaks in the coiled coil that may also form a central bubble; its absolute N-terminus is also likely splayed. The shorter Golgin-97 and GCC88 proteins are likely about half as long as GCC185 by sequence alone; Golgin-97’s N-terminus is likely splayed, and GCC88 may very well also contain a central bubble.

Finally, a domain between GCC185 residues 1332 and 1438 is required for maintenance of Golgi ribbon structure but not transport vesicle tethering (Brown et al., 2011). This observation highlights the fact that workers in this field have oversimplified thinking about how tethers function—simple engagement of N- and C-termini is not sufficient for both vesicle capture and Golgi ribbon stabilization. A recent paper studying Golgi tethers (Wong and Munro, 2014) concluded that unlike other TGN tethers, GCC185 could not tether vesicles upon re-localization to mitochondria. The extent of Golgi membrane mobilization seen in that study was far greater than the small pool size of the much rarer, Rab9-containing, AP-1 positive, mannose phosphate receptor transport vesicle intermediates—that are only visualized in cells upon depletion of tethering factors (Reddy, 2006; Brown et al., 2011). It is very possible that that study assayed the ability of TGN tethers to recruit/assemble TGN ribbons, rather than capture rare transport vesicle intermediates. Since ribbon assembly and transport vesicle capture are driven by separate domains of GCC185, perhaps GCC185 is more potent in vesicle tethering rather than Golgi ribbon assembly.

A remaining mystery is why depletion of any one of the GRIP domain-containing Golgins leads to Golgi fragmentation. Whatever the explanation, the floppy appearance of purified GCC185, when deposited on mica in the absence of fixative, and the proximity of GCC185’s N- and C-termini on the Golgi in cells, argue strongly against this protein behaving like a pipe cleaner, protruding on the surface of the trans Golgi network in cells. Instead, tether collapse represents an appealing, alternative model.

1. 1. Barbero P

   (2002) Visualization of Rab9-mediated vesicle transport from endosomes to the trans-golgi in living cells

   The Journal of Cell Biology 156:511–518.

   https://doi.org/10.1083/jcb.200109030

   • Google Scholar
2. 1. Barr FA
   2. Short B

   (2003) Golgins in the structure and dynamics of the golgi apparatus

   Current Opinion in Cell Biology 15:405–413.

   https://doi.org/10.1016/S0955-0674(03)00054-1

   • Google Scholar
3. 1. Brown FC
   2. Schindelhaim CH
   3. Pfeffer SR

   (2011) GCC185 plays independent roles in golgi structure maintenance and AP-1-mediated vesicle tethering

   The Journal of Cell Biology 194:779–787.

   https://doi.org/10.1083/jcb.201104019

   • Google Scholar
4. 1. Bröcker C
   2. Engelbrecht-Vandré S
   3. Ungermann C

   (2010) Multisubunit tethering complexes and their role in membrane fusion

   Current Biology 20:R943–R952.

   https://doi.org/10.1016/j.cub.2010.09.015

   • Google Scholar
5. 1. Burguete AS
   2. Fenn TD
   3. Brunger AT
   4. Pfeffer SR

   (2008) Rab and arl GTPase family members cooperate in the localization of the golgin GCC185

   Cell 132:286–298.

   https://doi.org/10.1016/j.cell.2007.11.048

   • Google Scholar
6. 1. Cai H
   2. Reinisch K
   3. Ferro-Novick S

   (2007) Coats, tethers, rabs, and SNAREs work together to mediate the intracellular destination of a transport vesicle

   Developmental Cell 12:671–682.

   https://doi.org/10.1016/j.devcel.2007.04.005

   • Google Scholar
7. 1. Carpenter AE
   2. Jones TR
   3. Lamprecht MR
   4. Clarke C
   5. Kang I
   6. Friman O
   7. Guertin DA
   8. Chang J
   9. Lindquist RA
   10. Moffat J
   11. Golland P
   12. Sabatini DM

   (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes

   Genome Biology 7:R100.

   https://doi.org/10.1186/gb-2006-7-10-r100

   • Google Scholar
8. 1. Derby MC
   2. Lieu ZZ
   3. Brown D
   4. Stow JL
   5. Goud B
   6. Gleeson PA

   (2007) The trans-golgi network golgin, GCC185, is required for endosome-to-golgi transport and maintenance of golgi structure

   Traffic 8:758–773.

   https://doi.org/10.1111/j.1600-0854.2007.00563.x

   • Google Scholar
9. 1. Derby MC

   (2004) Mammalian GRIP domain proteins differ in their membrane binding properties and are recruited to distinct domains of the TGN

   Journal of Cell Science 117:5865–5874.

   https://doi.org/10.1242/jcs.01497

   • Google Scholar
10. 1. Efimov A
    2. Kharitonov A
    3. Efimova N
    4. Loncarek J
    5. Miller PM
    6. Andreyeva N
    7. Gleeson P
    8. Galjart N
    9. Maia ARR
    10. McLeod IX
    11. Yates JR
    12. Maiato H
    13. Khodjakov A
    14. Akhmanova A
    15. Kaverina I

    (2007) Asymmetric CLASP-dependent nucleation of noncentrosomal microtubules at the trans-golgi network

    Developmental Cell 12:917–930.

    https://doi.org/10.1016/j.devcel.2007.04.002

    • Google Scholar
11. 1. Ganley IG
    2. Espinosa E
    3. Pfeffer SR

    (2008) A syntaxin 10 SNARE complex distinguishes two distinct transport routes from endosomes to the trans-golgi in human cells

    The Journal of Cell Biology 180:159–172.

    https://doi.org/10.1083/jcb.200707136

    • Google Scholar
12. 1. Hayes GL
    2. Brown FC
    3. Haas AK
    4. Nottingham RM
    5. Barr FA
    6. Pfeffer SR

    (2009) Multiple rab GTPase binding sites in GCC185 suggest a model for vesicle tethering at the trans-golgi

    Molecular Biology of the Cell 20:209–217.

    https://doi.org/10.1091/mbc.E08-07-0740

    • Google Scholar
13. 1. Houghton FJ
    2. Chew PL
    3. Lodeho S
    4. Goud B
    5. Gleeson PA

    (2009) The localization of the golgin GCC185 is independent of Rab6A/A' and Arl1

    Cell 138:787–794.

    https://doi.org/10.1016/j.cell.2009.05.048

    • Google Scholar
14. 1. Hughson FM
    2. Reinisch KM

    (2010) Structure and mechanism in membrane trafficking

    Current Opinion in Cell Biology 22:454–460.

    https://doi.org/10.1016/j.ceb.2010.03.011

    • Google Scholar
15. 1. Lin Y-C
    2. Chiang T-C
    3. Liu Y-T
    4. Tsai Y-T
    5. Jang L-T
    6. Lee F-JS

    (2011) ARL4A acts with GCC185 to modulate golgi complex organization

    Journal of Cell Science 124:4014–4026.

    https://doi.org/10.1242/jcs.086892

    • Google Scholar
16. 1. Lombardi D
    2. Soldati T
    3. Riederer MA
    4. Goda Y
    5. Zerial M
    6. Pfeffer SR

    (1993)

    Rab9 functions in transport between late endosomes and the trans golgi network

    The EMBO Journal 12:677–682.

    • Google Scholar
17. 1. Lupas A
    2. Van Dyke M
    3. Stock J

    (1991) Predicting coiled coils from protein sequences

    Science 252:1162–1164.

    https://doi.org/10.1126/science.252.5009.1162

    • Google Scholar
18. 1. Munro S

    (2011) The golgin coiled-coil proteins of the golgi apparatus

    Cold Spring Harbor Perspectives in Biology 3:a005256.

    https://doi.org/10.1101/cshperspect.a005256

    • Google Scholar
19. 1. Pfeffer SR

    (1999) Transport vesicle targeting: tethers before SNAREs

    Nature Cell Biology 1:E17.

    https://doi.org/10.1038/8967

    • Google Scholar
20. 1. Reddy JV

    (2006) A functional role for the GCC185 golgin in mannose 6-phosphate receptor recycling

    Molecular Biology of the Cell 17:4353–4363.

    https://doi.org/10.1091/mbc.E06-02-0153

    • Google Scholar
21. 1. Ren Y
    2. Yip CK
    3. Tripathi A
    4. Huie D
    5. Jeffrey PD
    6. Walz T
    7. Hughson FM

    (2009) A structure-based mechanism for vesicle capture by the multisubunit tethering complex Dsl1

    Cell 139:1119–1129.

    https://doi.org/10.1016/j.cell.2009.11.002

    • Google Scholar
22. 1. Sato K
    2. Roboti P
    3. Mironov AA
    4. Lowe M

    (2015) Coupling of vesicle tethering and rab binding is required for in vivo functionality of the golgin GMAP-210

    Molecular Biology of the Cell 26:537–553.

    https://doi.org/10.1091/mbc.E14-10-1450

    • Google Scholar
23. 1. Short B
    2. Haas A
    3. Barr FA

    (2005) Golgins and GTPases, giving identity and structure to the golgi apparatus

    Biochimica Et Biophysica Acta  1744:383–395.

    https://doi.org/10.1016/j.bbamcr.2005.02.001

    • Google Scholar
24. 1. Short B
    2. Preisinger C
    3. Korner R
    4. Kopajtich R
    5. Byron O
    6. Barr FA

    (2001) A GRASP55-rab2 effector complex linking golgi structure to membrane traffic

    The Journal of Cell Biology 155:877–884.

    https://doi.org/10.1083/jcb.200108079

    • Google Scholar
25. 1. Sinka R
    2. Gillingham AK
    3. Kondylis V
    4. Munro S

    (2008) Golgi coiled-coil proteins contain multiple binding sites for rab family g proteins

    The Journal of Cell Biology 183:607–615.

    https://doi.org/10.1083/jcb.200808018

    • Google Scholar
26. 1. Sztul E
    2. Lupashin V

    (2006) Role of tethering factors in secretory membrane traffic

    AJP: Cell Physiology 290:C11–C26.

    https://doi.org/10.1152/ajpcell.00293.2005

    • Google Scholar
27. 1. Wong M
    2. Munro S

    (2014) The specificity of vesicle traffic to the golgi is encoded in the golgin coiled-coil proteins

    Science 346:1256898.

    https://doi.org/10.1126/science.1256898

    • Google Scholar
28. 1. Yamakawa H
    2. Seog D-H
    3. Yoda K
    4. Yamasaki M
    5. Wakabayashi T

    (1996) Uso1 protein is a dimer with two globular heads and a long coiled-coil tail

    Journal of Structural Biology 116:356–365.

    https://doi.org/10.1006/jsbi.1996.0053

    • Google Scholar
29. 1. Yu I-M
    2. Hughson FM

    (2010) Tethering factors as organizers of intracellular vesicular traffic

    Annual Review of Cell and Developmental Biology 26:137–156.

    https://doi.org/10.1146/annurev.cellbio.042308.113327

    • Google Scholar

Author details

1. Pak-yan Patricia Cheung

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   PyanPC, Carried out the biochemical and cell biology experiments, discussed the results and wrote and/or commented on the final manuscript

   Competing interests

   No competing interests declared.

2. Charles Limouse

   Department of Applied Physics, Stanford University, Stanford, United States

   Contribution

   CL, Carried out the AFM and was key to the analysis of AFM data, discussed the results and wrote and/or commented on the final manuscript

   Competing interests

   No competing interests declared.

3. Hideo Mabuchi

   Department of Applied Physics, Stanford University, Stanford, United States

   Contribution

   HM, Discussed the results and wrote and/or commented on the final manuscript

   Competing interests

   No competing interests declared.

4. Suzanne R Pfeffer

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   SRP, Contributed to the study design, discussed the results and wrote and/or commented on the final manuscript

   For correspondence

   pfeffer@stanford.edu

   Competing interests

   SRP: Reviewing editor, eLife.

   "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

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