VAMP3/Syb and YKT6 are required for the fusion of constitutive secretory carriers with the plasma membrane

The cellular machinery required for the fusion of constitutive secretory vesicles with the plasma membrane in metazoans remains poorly defined. To address this problem we have developed a powerful, quantitative assay for measuring secretion and used it in combination with combinatorial gene depletion studies in Drosophila cells. This has allowed us to identify at least three SNARE complexes mediating Golgi to PM transport (STX1, SNAP24/29 and Syb; STX1, SNAP24/29 and YKT6; STX4, SNAP24 and Syb). RNAi mediated depletion of YKT6 and VAMP3 in mammalian cells also blocks constitutive secretion suggesting that YKT6 has an evolutionarily conserved role in this process. The unexpected role of YKT6 in plasma membrane fusion may in part explain why RNAi and gene disruption studies have failed to produce the expected phenotypes in higher eukaryotes

SNAP24 and SNAP29 mediate the fusion of secretory carriers with the plasma membrane.

<p>A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with DD solubiliser at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for three repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with DD solubiliser at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting the indicated genes. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.</p

R-SNARE homologues in different organisms.

<p>R-SNARE homologues in different organisms.</p

STX1, STX4 and Syb are required for the fusion of secretory carriers with the plasma membrane.

<p>A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). *The apparent increase in STX1 and STX4 levels by immunoblotting when STX5 and Syb are depleted is not because of a change in total STX1 levels but is due to a change in its extractability from cells. No difference in STX1 or STX4 levels were observed when cells are directly prepared in Laemmli sample buffer (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006698#pgen.1006698.s002" target="_blank">S2 Fig</a>). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX1 and STX4. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.</p

Genetic interactions support a role for YKT6 in the mammalian late secretory pathway.

<p>A) Representative histograms from a secretion experiment. Clone 1 cells were mock transfected (Oligofectamine only) or transfected with siRNA targeting the indicated genes. After 96 hours, the cells were incubated with D/D solubiliser at 37°C for 80 minutes and their mean fluorescence determined using flow cytometry. The red histogram shows the fluorescent intensity of the control sample, no DD solubiliser and the blue histogram shows the fluorescent intensity of the cells incubated with DD solubiliser. The amount of cargo remaining in the cells was calculated and plotted in B (Error bars show experimental standard deviation for three repeats).</p

YKT6 functions on multiple intracellular pathways.

<p>A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting YKT6 and Sec22b. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.</p

Development of a novel assay for measuring secretion in <i>Drosophila</i> cells.

<p>A) Schematic of the reporter construct used to measure secretion (DD, dimerisation domain; FCS, furin cleave site; hGH, human growth hormone and numbers indicate amino acids). B) Transport kinetics of the reporter construct were determined by incubating C3 cells with AP21998 at 25°C for the indicated times and the mean fluorescence of the cells measured using flow cytometry. The amount of cargo remaining in the cells after the addition of AP21998 was calculated as a ratio between the control sample (no AP21998) and the experimental samples (+AP21998). C) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The red histogram indicates the fluorescent intensity of the control sample, no AP21998 and the blue histogram shows the fluorescent intensity of the cells incubated with AP21998. D) The amount of cargo remaining in the cells was calculated as in B and plotted (Error bars show experimental range for three repeats). E) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. F) Clone 3 cells were either mock transfected or transfected with dsRNA targeting STX5 and ROP. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm.</p

Syb and YKT6 have a role in the fusion of secretory carriers with the plasma membrane.

<p>A) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and their mean fluorescence determined using flow cytometry. The amount of cargo remaining in the cells was calculated and plotted (Error bars show experimental range for six repeats). B) Clone 3 cells were mock transfected (TransFast only) or transfected with dsRNA targeting the indicated genes. After 96 hours, the cells were incubated with AP21998 at 25°C for 80 minutes and the media and cells harvested for immunoblotting. Solid arrowhead indicates unprocessed cargo and unfilled arrowhead furin processed cargo. The amount of processed GH in the media was quantified using densitometry and plotted in C (Error bars show experimental range for two repeats). D) Clone 3 cells were either mock transfected or transfected with dsRNA targeting YKT6 and Syb. After 72 hours the cells were seeded onto coverslips. The next day, the cells were incubated with AP21998 at 25°C for 80 minutes. The cells were then fixed and stained for the Golgi marker GM130. Scale Bar 10μm. E) To determine which SNAREs interact with STX1 a native immunoprecipitation was performed from <i>Drosophila</i> cells stably expressing HA tagged STX1. The isolated protein complexes were separated by SDS-PAGE and the major bands identified using mass-spectrometry.</p

Proteins identified co-immunoprecipitating with STX1.

<p>Proteins identified co-immunoprecipitating with STX1.</p

At least three SNARE complexes function in the fusion of secretory vesicles with the <i>Drosophila</i> plasma membrane

<p>A) Diagram summarising the functional and biochemical data presented in this study. Our data supports the role of at least three SNARE complexes acting at the plasma membrane in <i>Drosophila</i> cells. B) Diagram summarising the <i>Drosophila</i> genetic interactions in relation to published <i>S</i>. <i>cerevisiae</i> interaction data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006698#pgen.1006698.ref058" target="_blank">58</a>]. Figure reprinted with permission of the authors. S-Scores were calculated based on colony growth of double mutants as previously described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006698#pgen.1006698.ref063" target="_blank">63</a>]. Blue indicates negative genetic interactions, black indicates no/neutral genetic interactions |S-score| < 0.5, and grey indicates no data for this gene combination (ND). To characterize essential genes in yeast, a strategy known as decreased abundance by mRNA perturbation (DAmP) was used to generate hypomorphic strains (analogous to gene knockdown). A |S-score| > 3 generally indicates a high confidence genetic interaction.</p