Golgin45-Syntaxin5 Interaction Contributes to Structural Integrity of the Golgi Stack

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.

Helical wheel plots showing the positions of homologous amino acid residues found in the leucine zipper-like motifs of GM130 and Golgin45. Boxed residues indicate identical or similar AA residues; (B) Alanine scanning mutagenesis of common AA residues within Leucine zipper motif of Golgin45 and GST pull-down assays were used to identify a specific point mutant of Golgin45 that fails to bind recombinant Syntaxin5. D171A mutation significantly abrogates Golgin45 CC domain binding to GST-Syntaxin5 (1-215) in pull-down assays. (C) GST-Rab2.Q65L recombinant proteins were used to test if Golgin45.D171A mutant show any change in their interaction, compared to the WT Golgin45. Both the WT and D171A.Golgin45 equally bound to Rab2 in GTP-dependent manner, suggesting Syntaxin5-binding deficient mutant is not affected in Rab2 interaction in vitro. (D) Expression of mCherry-Golgin45.D171A does not significantly affect secretion of soluble secretory cargo, ss-HRP, in HeLa cells, compared to the cells transfected with vector-transfected control, while expression (2019) 9:12465 | https://doi.org/10.1038/s41598-019-48875-x www.nature.com/scientificreports www.nature.com/scientificreports/ 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 directly 15 , 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 Syntaxin5 directly.

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 (CC1-3), 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 characterize 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 observed phenotype.
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 cells 11 . 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 hours 11 . 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 study 11 . 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. www.nature.com/scientificreports www.nature.com/scientificreports/ EM tomography further confirms inter-cisternal fusion, induced by expression of the Golgin45 D171A mutant. To further support these findings, we performed EM tomography to obtain higher quality images of fused Golgi cisterna at EM resolution. HeLa cells were treated with GRASP65/55 siRNAs, followed by expression of the mCherry-Golgin45 D171A mutant for 18 hours. The EM tomograph images from these experiments indeed confirmed that there are extensive intercisternal fusions throughout the Golgi stacks in these cells ( Fig. 4B; movie #1). Taken together, these results indicate that Golgin45-Syntaxin5 interaction may have a role in structural integrity of the Golgi stack.
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  www.nature.com/scientificreports www.nature.com/scientificreports/ 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. www.nature.com/scientificreports www.nature.com/scientificreports/ 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 stacks 20 , as are one or another of Golgin45 and GM130 3,5 . 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.
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 pC4-mCherry (vector control), pC4-mCherry-Golgin45 WT or D171A mutant plasmids, respectively, using