Golgin45-Syntaxin5 Interaction Contributes to Structural Integrity of the Golgi Stack
Golgin45-Syntaxin5 Interaction Contributes to Structural Integrity of the Golgi Stack
James E. Rothman
The unique stacked morphology of the Golgi apparatus had been a topic of intense investigation among the cell biologists over the years. We had previously shown that the two Golgin tethers (GM130 and Golgin45) could, to a large degree, functionally substitute for GRASP-type Golgi stacking proteins to sustain normal Golgi morphology and function in GRASP65/55-double depleted HeLa cells. However, compared to well-studied GM130, the exact role of Golgin45 in Golgi structure remains poorly understood. In this study, we aimed to further characterize the functional role of Golgin45 in Golgi structure and identified Golgin45 as a novel Syntaxin5-binding protein. Based primarily on a sequence homology between Golgin45 and GM130, we found that a leucine zipper-like motif in the central coiled-coil region of Golgin45 appears to serve as a Syntaxin5 binding domain. Mutagenesis study of this conserved domain in Golgin45 showed that a point mutation (D171A) can abrogate the interaction between Golgin45 and Syntaxin5 in pull-down assays using recombinant proteins, whereas this mutant Golgin45 binding to Rab2-GTP was unaffected in vitro. Strikingly, exogenous expression of this Syntaxin5 binding deficient mutant (D171A) of Golgin45 in HeLa cells resulted in frequent intercisternal fusion among neighboring Golgi cisterna, as readily observed by EM and EM tomography. Further, double depletion of the two Syntaxin5-binding Golgin tethers also led to significant intercisternal fusion, while double depletion of GRASP65/55 didn't lead to this phenotype. These results suggest that certain tether-SNARE interaction within Golgi stack may play a role in inhibiting intercisternal fusion among neighboring cisternae, thereby contributing to structural integrity of the Golgi stack.
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The Golgi apparatus is a central sorting station that orchestrates membrane transport at the cross-road of the
secretory and the endocytic pathways1. The biogenesis of the Golgi apparatus is known to be mediated by a group
of Golgin tethers and two Golgi Reassembly and Stacking proteins (GRASPs; GRASP65/55), and the Golgi
undergoes dynamic disassembly and reassembly during mitotic cell division2–6. GRASPs have been shown to mediate
Golgi stacking by their PDZ domain interaction in trans7,8. On the other hand, Golgins, such as GM130, seem
to be essential for SNARE-mediated membrane fusion of mitotic Golgi membranes for regeneration of Golgi
cisternae during post-mitotic Golgi reassembly9,10. However, it is unclear by what mechanism GM130 directly
contributes to cisternal stacking.
We recently reported that both GM130 and Golgin45 can substitute for GRASPs to such an extent that their
exogenous over-expression can create functionally normal Golgi stacks in GRASP65/55-depleted mammalian
cells, suggesting that the two Golgins and GRASPs play extensively complementary roles under physiological
conditions11. This is consistent with the findings that GRASP homologs, Grh1 and dGRASP, are each largely
dispensable for Golgi stacking in yeast S.pombe and drosophila S2 cells, respectively12,13.
GM130 utilizes its N-terminal domain (proximal to its Cdc2 phosphorylation site) to bind to another Golgin
tether, p11514,15 and to Golgi tSNARE Syntaxin5 via its coiled-coil domain 4–6 (CC4-6)15. GM130 also interact
directly with Rab1-GTP (using CC1-3) and Rab33b-GTP (CC4-6)16–18. These interactions are known to play
important roles in coordinating SNARE-mediated membrane fusion of incoming ER-derived cargo carriers
to cis-Golgi cisternae17, but precise binding interactions of Syntaxin5 and these Rab-GTPases with GM130 are
poorly understood15. Golgin45 is known to bind Rab2-GTP and ACBD3 via its central coiled-coil region and
GRASP55 via its C-terminal PDZ-binding motif3,7,19, but the exact mechanism by which Golgin45 contributes to
Golgi structure has remained elusive.
In this study, we report a novel interaction between Golgin45 and a Golgi tSNARE, Syntaxin5. We further
demonstrate using EM and EM tomography that this protein-protein interaction between the two Golgi matrix
components significantly contributes to structural integrity of the Golgi stack by inhibiting intercisternal fusion
between neighboring Golgi cisterna.
Results and Discussion
Golgin45 is a syntaxin5-binding Golgin tether. In order to characterize the functional role of Golgin45
on Golgi structure, we first postulated that Golgin45 could be a functional homologue of GM130, based on its
interaction with GRASP55 (GRASP65 for GM130) and Rab2-GTP (Rab33b-GTP for GM130). This assumption
of mCherry-Golgin45 WT very moderately inhibits secretion of ss-HRP. (E) D171A mutation does not affect
Golgin45 targeting to the Golgi and its co-localization with endogenous GRASP55 in HeLa cells, compared to
that of Golgin45 WT. Line analysis graph shows that both the WT and the mutant mCherry-Golgin45.D171A
co-localize well with anti-GRASP55 stained Golgi area. Bar = 10 μm.
led us to hypothesize that, like GM130, Golgin45 may also bind Golgi tSNARE, Syntaxin5, using its Rab-binding
domain. Upon close examination of amino acid sequence homology, we found highly conserved leucine
zipper-like motifs, commonly shared by GM130 (CC5) and Golgin45 (CC2) (Fig. 1A,B). As this region of GM130
(CC4-6) had previously been implicated in binding Syntaxin5 directly15, we posited that CC2 of Golgin45 could
interact with Syntaxin5 as well. Initially, we used GST pull-down assays to test whether Syntaxin5 can bind
both GM130 and Golgin45 from HeLa cell extract. The results showed (Fig. 1C) that GST-Syntaxin5, but not
GST-GS15, captured GM130 and YFP-Golgin45 from HeLa cell extract, suggesting that both Golgins might bind
N-terminal regulatory domain (H3) of Syntaxin5 interacts with Golgin45. As individual CC
domains of both GM130 and Golgin45 were highly unstable in solution upon purification, 6xHis-tagged
recombinant GM130 (CC4-6) and Golgin45 (CC1-3) were expressed and purified from BL21 to further study their
binding interaction to Syntaxin5 and its truncation mutants. The results showed that the N-terminal regulatory
domain (H3) of Syntaxin5 (amino acids (AA) 1-215) is likely to be responsible for its binding to Golgin45
(CC13), whereas the SNARE domain (AA215-275) failed to show any binding (Fig. 1D,E). Binding of recombinant
GM130 (CC4-6) to GST-Syntaxin5 showed a similar pattern (Fig. 1F), suggesting that GM130 and Golgin45 seem
to use the leucine zipper-like domain for binding to Syntaxin5 H3 domain.
The D171A mutation in Golgin45 CC2 abrogates its binding to syntaxin5. To further character
ize the binding interaction, alanine scanning mutagenesis of Golgin45 CC2 was carried out to identify a point
mutation that abrogates Golgin45-Syntaxin5 interaction (Fig. 2A; see boxed amino acid residues in the helical
wheel plots). We then performed GST pull-down assays using the purified recombinant mutant proteins and
GST-Syntaxin5 N-terminal domain (
). The results showed that D171A mutation causes a significant
reduction in Golgin45-Syntaxin5 binding, but not in the interaction between Rab2-GTP and Golgin45 (Fig. 2B,C).
To examine the effect of this mutation on Golgi function, we co-transfected HeLa cells with a soluble secretory
cargo (ss-HRP) and either mCherry tagged Golgin45 WT or Golgin45.D171A mutant for 18 hours. We then used
ELISA assays to detect secreted ss-HRP in the conditioned media. The results (Fig. 2D) showed that expression of
either mCherry-Golgin45 WT or the mutant Golgin45 resulted in moderate reduction in the amount of ss-HRP
in the conditioned media, compared to vector-transfected control cells, suggesting that anterograde protein
secretions are not significantly affected in these cells.
Confocal results suggested that both mCherry-Golgin45 WT and the mutant mCherry-Golgin45.D171A were
correctly targeted to the Golgi and co-localized well with endogenous GRASP55 (Fig. 2E), demonstrating that
Syntaxin5 binding may not be important for Golgin45 targeting to the Golgi.
Expression of the Golgin45 D171A mutant results in inter-cisternal fusion. In order to test
whether expression of the Golgin45 D171A mutant might influence Syntaxin5 localization to the Golgi, we
transfected mCherry-tagged Golgin45 WT or D171A mutant in HeLa cells for 18 hours. The cells were then fixed and
stained with anti-GM130 (Golgi marker) and anti-Syntaxin5 antibodies for examination under confocal
microscope. The results (Fig. 3A) showed that expression of Golgin45.D171A mutant had no obvious effect on Golgi
structure and Syntaxin5 localization to the Golgi at light microscope level.
These cells were then processed for EM to further study any alteration of the Golgi structure at EM
resolution. Strikingly, we found that exogenous expression of the D171A mutant, but not the WT Golgin45, resulted
in frequent intercisternal fusions among Golgi cisterna (Fig. 3B), suggesting that this tether-SNARE interaction
may be important for structural integrity of the Golgi stack, although we cannot rule out the possibility that this
particular mutation (D171A) may disrupt some other important protein-protein interaction(s), leading to the
The Golgin45 D171A mutant fails to restore normal Golgi morphology in GRASP-double depleted
heLa cells. We had previously demonstrated that exogenous expression of WT Golgin45 restores normal
Golgi stack morphology and secretory function in GRASP65/55 double-depleted cells11. We used this experiment
as an assay to test the hypothesis that, if Golgin45-Syntaxin5 interaction is crucial for Golgi structural integrity,
the mutant Golgin45 (D171A) may fail to restore normal Golgi morphology in GRASPs double-depleted cells.
We treated HeLa cells with siRNAs against human GRASP65 and GRASP55 for 48 hours, followed by transfection
with either mCherry-Golgin45 WT (control) or mCherry-Golgin45 D171A mutant for 18 hours11. The cells were
then processed for electron microscopy.
The results showed that, as expected, exogenous expression of WT Golgin45 restored normal Golgi stack
morphology (Fig. 4A; bottom left panel) to a significant extent in these cells, although the cisterna in the restored
Golgi were still moderately dilated and the Golgi ribbon remained fragmented. However, expression of the
D171A mutant not only failed to restore Golgi morphology, but also resulted in frequent intercisternal fusion
among the severely dilated Golgi cisterna (Fig. 4A; bottom right panel). Due to significant membrane fusion
among the neighboring cisternae, it was difficult to quantify this change by measuring maximum cisternal width,
as was done in our previous study11.
Individual knock-down of Golgin45 or GRASP55 leads to a reduction in the average number
of cisternae per Golgi stack but does not result in inter-cisternal fusion. Interestingly, we failed
to observe intercisternal fusion in HeLa cells treated with Golgin45 siRNAs for 96 hours. Nor did we find
intercisternal fusion in GM130 or GRASP-depleted cells (Fig. 5A–E). However, we did find significant reduction in the
average number of cisternae per stack in Golgin45 KD cells and GRASP55 KD cells (Fig. 5F,G; also see histograms
in Supplementary Fig. 1), suggesting that these two medial Golgi stacking proteins may play the most critical role
for Golgi structure.
Simultaneous depletion of the two Syntaxin5-binding Golgins results in inter-cisternal fusion.
As these Golgins and GRASP-type proteins seem to be capable of functionally substituting for one another, we then
decided to examine the effect of Golgin double KD or GRASP65/55 double KD to see whether double depletion of
either the two Syntaxin5-binding Golgins or the GRASPs may lead to inter-cisternal fusion. Interestingly,
simultaneous depletion of GM130/Golgin45 (Fig. 6) resulted in massive fusion among 3–4 neighboring cisterna, whereas
neither the control siRNA-treated nor GRASP65/55 depleted cells (see Fig. 4A; upper panel) resulted in this phenotype.
We routinely found various degrees of inter-cisternal fusion in ~20% of GM130/Golgin45 double-depleted
HeLa cells examined under EM. Again, it was difficult to systematically quantify inter-cisternal fusion, partly due
to highly diverse morphologies of the fused Golgi membranes on these EM photos, which were obtained using
thin section EM method that was employed for these experiments.
Syntaxin5 is known to be localized throughout the stacks20, as are one or another of Golgin45 and GM1303,5.
Therefore, this binding interaction may help prevent inter-cisternal fusion through a significant portion of the
Golgi stack. In summary, we propose that Golgin45-Syntaxin5 interaction represents a novel protein-protein
interaction among the Golgi matrix components, which may contribute to structural integrity of the Golgi stack.
Reagents and antibodies. All common reagents were purchased from Sigma-Aldrich (St.Louis, MO),
unless otherwise mentioned. The following antibodies were used: mouse monoclonal GM130 (BD Transduction
Laboratories), goat polyclonal GRASP65 (Santa Cruz Biotechnology, CA), rabbit polyclonal GRASP55
(Proteintech inc., Chicago,IL), HRP-conjugated mouse β-actin antibody (Genscript, Piscataway, NJ). Rabbit
polyclonal Syntaxin5 (Synaptic system, Goettingen, Germany). Rabbit polyclonal Golgin45 antibody was made by
injecting synthetic Golgin45 peptide (AA40-53) conjugated to KLH (GenScript, Piscataway, NJ). All siRNA oligos
were purchased from Integrated DNA Technology (Coralville, IA) and the target sequences were as following:
human Golgin45 (GCATCATAGTCTTCAGAGTCCATGG), Human Golgin45 (5′UTR for rescue experiments)
(CGGAGAAUAAGAAUCUUAGAGGU), Human GM130 (GGACAATGCTGCTACTCTACAACCA), GRASP55
oligo#1 (CTGCGAGAGACCTCAGTCACACCAA), GRASP55 oligo#2 (CCACCAGGAACTACAGGAAT
TGAAC), GRASP65 oligo#1 (CCTGAAGGCACTACTGAAAGCCAAT), GRASP65 oligo#2 (CTGGGATGTGGC
ATTGGCT ATGGGT). Rat GM130 cDNA was obtained from Nobuhiro Nakamura (Kyoto Sangyo University,
Kyoto, Japan). Human Golgin45 cDNA was purchased from Addgene (Cambridge,MA).
Cell culture and treatments. HeLa cells were grown in DMEM supplemented with 10% FBS (Invitrogen,
Carlsbad, CA) at 37 C. For transfection with siRNA, cells were plated onto 6-well plates 24 hours prior to
transfection. We performed the transfection using Dharmafect-1 (HeLa) (Dharmacon, Boulder, CO), according to
manufacturer’s instruction. cDNA transfection was done using standard protocol using Lipofectamine2000 (Invitrogen,
Carlsbad, CA) or FugeneHD (Promega, Sunnyvale, CA). Confocal images were obtained using LSM880 confocal
microscope (Carl Zeiss, Dublin, CA).
Immunofluorescence staining. Cells were fixed in 4% PFA in PBS for 15 min at room temperature (RT),
followed by permeabilization in 0.3% Triton X-100 in PBS for 3 min. After 3 times washing with PBS, cells were
then blocked in blocking buffer containing 2% BSA, 0.05% Triton X-100 in PBS for 30 min at RT. Primary
antibodies were diluted in blocking buffer, according to manufacturer’s instruction and incubated with cells for
30 min at RT. Secondary antibodies conjugated to Alexa dyes were diluted in blocking buffer and incubated with
cells for 15 min at RT.
SS-HRP secretion assay. HeLa cells were co-transfected with pcDNA3.1-ss-HRP plasmid plus
pC4mCherry (vector control), pC4-mCherry-Golgin45 WT or D171A mutant plasmids, respectively, using
Lipofectamine 3000 (Life Technologies). The conditioned media were harvested 18hours post-transfection. HRP
activity was measured using 1-Step Ultra TMB-ELISA (Thermo), according to the manufacturer’s instructions.
Experiments were carried out in triplicate samples and repeated twice.
GST pull-down assays. Recombinant 6xHis-T7-tagged Golgin45 and GM130 CC domains were expressed
in BL21 and purified using manufacturer’s standard protocols. We found that GST-rat Syntaxin5 constructs were
highly insoluble in various detergent and salt conditions, and eventually opted to use 2% sarkosyl in TBS and
probe sonication to break up the aggregated recombinant proteins in the inclusion bodies and diluted the lysates
up to 10-fold in protein refolding buffer containing 2% Triton-X 100 and 30 mM CHAPS plus protease inhibitor
cocktail (Roche) in TBS, prior to binding to Glutathione sepharose for purification. Typically, we used 50–100nM
recombinant CC domain proteins and incubated with 50 μg purfied GST-Syntaxin5 on Glutathione beads for
3 hrs at 4 C.
Image analysis on electron micrographs and data presentation. Unless otherwise stated, we used
digital electron micrographs in Tiff format and ImageJ software (NIH) to study the number of cisternae per Golgi
stack and cisternal luminal width measurement. To comprehensively assess the functional relationship of the
GRASPs and the Golgins, a large number of cisternae and stacks in numerous sections were studied extensively.
We performed two independent experiments for each dataset in the figures. Frequency distribution histograms
from these experiments are shown in Supplementary Fig. 1.
Sample preparation and image acquisition for electron microscopy/tomography. The trans
fected cells were fixed in 2.5% gluteraldehyde in 0.1 M sodium cacodylate buffer pH7.4 with for 1 hour. They
were then rinsed in 0.1 M sodium cacodylate buffer, scraped and pelleted in 2% agar. Samples were trimmed
and post-fixed in 1% osmium tetroxide for 1 hour, en bloc stained in 2% uranyl acetate in maleate buffer pH5.2
for a further hour, rinsed then dehydrated in an ethanol series and infiltrated with resin (Embed812 Electron
Microscopy Science) and baked over night at 60 C. Hardened blocked were cut using a Leica UltraCut UC7,
60 nm sections were collected onto formvar/carbon coated nickel grids and stained using 2% uranyl acetate and
lead citrate. These were viewed FEI Tecnai Biotwin TEM at 80 Kv. Images were taken using Morada CCD and
iTEM (Olympus) software typically at 26,000 x magnification. For electron tomography, 250 nm sections were
collected on formvar/carbon copper grids, labeled on both sides with 10 nm gold particles (UtrectUMC). A
tomography tilt series was acquired using FEI Express 3D software on an FEI Tecnai TF20 FEG TEM at 200 kV.
Images were reconstructed using IMOD software (University of Colorado, Boulder, CO).
The authors thank Stuart Kornfeld for critical reading of the manuscript and Mijeong Kim for the animated
illustrations of the EM tomographic results. The early stages of this research were carried out at Yale by I.L. when
he was a postdoctoral fellow in J.E.R.’s laboratory at which time the research was supported by an NIH from G.M.
to J.E.R. The work was then completed by I.L. independently at ShanghaiTech without financial support from NIH.
Competing Interests: The authors declare no competing interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-48875-x.