Traffic 2010; 11: 1191–1204 © 2010 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2010.01087.x

A Targeted siRNA Screen to Identify SNAREs Requiredfor Constitutive Secretion in Mammalian Cells

David E. Gordon, Lisa M. Bond, Daniela

A. Sahlender and Andrew A. Peden∗

Department of Clinical Biochemistry, University ofCambridge, Cambridge Institute for Medical Research,Wellcome Trust/MRC Building, Hills Road, CambridgeCB20XY, UK*Corresponding author: Andrew A. Peden,aap20@cam.ac.uk

The role of SNAREs in mammalian constitutive secretion

remains poorly defined. To address this, we have devel-

oped a novel flow cytometry-based assay for measuring

constitutive secretion and have performed a targeted

SNARE and Sec1/Munc18 (SM) protein-specific siRNA

screen (38 SNAREs, 4 SNARE-like proteins and 7 SM

proteins). We have identified the endoplasmic reticulum

(ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18,

GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein

Sly1, along with the post-Golgi SNAREs SNAP-29 and

syntaxin 19, as being required for constitutive secretion.

Depletion of SNAP-29 or syntaxin 19 causes a decrease

in the number of fusion events at the cell surface and in

SNAP-29-depleted cells causes an increase in the number

of docked vesicles at the plasma membrane as deter-

mined by total internal reflection fluorescence (TIRF)

microscopy. Analysis of syntaxin 19-interacting partners

by mass spectrometry indicates that syntaxin 19 can form

SNARE complexes with SNAP-23, SNAP-25, SNAP-29,

VAMP3 and VAMP8, supporting its role in Golgi to plasma

membrane transport or fusion. Surprisingly, we have

failed to detect any requirement for a post-Golgi-specific

R-SNARE in this process.

Key words: assay, CEDNIK, constitutive secretion, SNAP-

29, SNARE, syntaxin, syntaxin 19, TIRF, VAMP

Received 4 May 2010, revised and accepted for publi-

cation 8 June 2010, uncorrected manuscript published

online 10 June 2010, published online 7 July 2010

Constitutive secretion is an essential, conserved processthat is required for the delivery of newly synthesizedproteins and lipids to the plasma membrane (PM) andthe exocytosis of extracellular factors (1). Through acombination of yeast genetics, cloning and biochemicalapproaches, many of the core components of this pathwayhave been identified. This includes machinery required forvesicle biogenesis [coat protein II (COPII)], vesicle tether-ing (exocyst) and vesicle fusion (SNAREs) (2–4).

SNAREs are generally small (14–40 kDa), coiled-coilforming proteins that are anchored to the membrane via

a C-terminal anchor (5–7). For fusion to occur, SNAREson opposing membranes must come together (7) andform a parallel four-helical bundle (8). It is thought thatthe zippering of the coiled-coil domains brings the mem-branes in close enough proximity to drive bilayer fusion.The coiled-coil domains of SNAREs are highly con-served allowing them to be subdivided into two mainclasses, R-SNAREs that have an arginine at the centreof their coiled-coil domain and Q-SNAREs that have aglutamine (9).

In Saccharomyces cerevisiae, all of the SNAREs requiredfor constitutive secretion have been identified and char-acterized in great detail (10). The Q-SNAREs Sed5, Bos1,Bet1 and the R-SNAREs Sec22 or Ykt6 are required forthe anterograde transport of cargo between the endo-plasmic reticulum (ER) and the Golgi. The Q-SNAREsSso1 or Sso2, Sec9 or Spo20 and the R-SNAREs Snc1or Snc2 are required for fusion of post-Golgi vesicleswith the PM. In mammalian cells, the SNAREs requiredfor the ER-to-Golgi transport are also well defined andconsist of the Q-SNAREs syntaxin 5, GS27, Bet1 andthe R-SNAREs Sec22b or Ykt6 (11). However, it stillremains unclear which SNAREs are required for fusionof Golgi-derived vesicles with the PM. The most likelySNAREs to be involved in this process are syntaxins 1A,1B, 2, 3 and 4 (homologues of Sso1), SNAP-23, 25, 29 and47 (homologues of Sec9) and VAMPs 1, 2, 3, 4, 5, 7 and 8(homologues of Snc1). Attempts to identify which of theseSNAREs are required for fusion of Golgi-derived vesicleswith the PM so far have been unsuccessful (12,13).

To identify which SNAREs are required for constitutivesecretion in mammalian cells, we have developed anovel flow cytometry-based assay for measuring con-stitutive secretion and used this in combination withan siRNA-based screening approach. We have assayedevery annotated SNARE and SM protein in the humangenome (14,15) and identified the ER/Golgi SNAREs syn-taxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20,Sec22b, YKT6 and the SM protein Sly1, along with thepost-Golgi SNAREs SNAP-29 and syntaxin 19, as beingrequired for constitutive secretion. Depletion of SNAP-29and syntaxin 19 reduces the number of vesicles fusingwith the PM and in SNAP-29-depleted cells causes anaccumulation of docked vesicles under the cell surface.We have also shown that haemagglutinin (HA)-taggedsyntaxin 19 is able to form SNARE complexes with SNAP-23, 25 and 29 as well as VAMPs 3 and 8, indicatinga role in post-Golgi trafficking or fusion with the PM.Surprisingly, we have failed to detect any requirementfor a post-Golgi-specific R-SNARE in the secretion of thereporter construct.

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Results

The development of a flow cytometry-based assay

for measuring constitutive secretion

To quantitatively measure constitutive secretion in mam-malian cells, we have made use of the Ariad RPD Reg-ulated Secretion/Aggregation Kit (16). This kit makes useof a reporter construct that is based around the propertyof mutant FKBP proteins (F36M) to form ligand-reversibledimers. When mutant FKBP proteins are linked together,they can form large aggregates that when expressed inthe ER cannot be secreted. However, when cells areincubated with the FKBP (F36M) ligand AP21998, theseaggregates are solubilized and a synchronous pulse of

reporter construct is efficiently and rapidly secreted fromthe cells (17,18). We have modified the reporter constructincluded in the kit so that it contains a green fluorescentprotein (eGFP) tag, allowing for secretion to be quantifiedusing optical-based approaches (Figure 1A). Initial experi-ments using the reporter construct transiently did not givesatisfactory results so a stable clonal cell line (C1) wasgenerated.

Addition of AP21998 to C1 cells causes a significantreduction in the cells’ fluorescence as the reporter con-struct is secreted. Thus, we realized that this changein fluorescence could be the basis of a simple, direct,quantitative assay for measuring constitutive secretion.

Figure 1: Development of a flow cytometry-based assay for measuring constitutive secretion. A) Schematic of the reporterconstruct used to measure secretion (SS, signal sequence; eGFP, enhanced green fluorescent protein; FM, FKBP mutated; FCS, furincleavage sequence; hGH, human growth hormone and numbers represent amino acids). B) To measure the time–course for secretionof the reporter constructs, C1 cells were incubated with AP21998 (1 μM) for the indicated times at 37◦C and their mean fluorescencedetermined using flow cytometry (graph) and the appearance of reporter construct in the media determined by immunoblotting (blot).The percentage of construct remaining in the cells after the addition of AP21998 was calculated as a ratio between a control sample (noAP21998) and the experimental samples (+AP21998). C) To determine if the secretion of the reporter construct was brefeldin A-sensitive,C1 cells were incubated either in the presence or absence of brefeldin A (2 μg/mL) and AP21998 (1 μM) for 80 min at 37◦C and theirmean fluorescence determined using flow cytometry. Error bars represent the experimental range of three repeats. D) To determine ifthe secretion of the reporter construct was COPII dependent, C1 cells were transfected with either CFP or dominant-negative CFP-Sar1(H79G) for 48 hours and then incubated with AP21998 (1 μM) for 80 min at 37◦C and their mean fluorescence determined using flowcytometry. Error bars represent the experimental range of three repeats.

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To test this hypothesis, a time–course experiment wasperformed where C1 cells were incubated with 1 μM

AP21998 over a period of 100 min at 37◦C. The fluo-rescence of the cells was then measured using flowcytometry (Figure 1B, graph) and the media collected fromthe cells to determine if the construct was being secretedinto the media (Figure 1B, blot). The fluorescence of theC1 cells decreased significantly over the time–course ofthe experiment and this correlates with the appearanceof the reporter construct (GFP and growth hormone (GH))in the media. As the reporter construct contains a furincleavage site, a significant amount of the construct hasbeen processed indicating that the construct has traffickedthrough the trans Golgi network (TGN). The presence ofunprocessed construct (101-kDa band) in the media ismost likely caused by saturation of this process.

To confirm that the loss of fluorescence in the C1 cells wasbecause of the secretion of the reporter construct and todetermine that the reporter construct was being secretedusing the conventional secretory pathway, we have testedthe effect of two treatments known to block constitutivesecretion, brefeldin A that perturbs Golgi morphologyand overexpression of a dominant-negative Sar1 mutant(H79G) that inhibits COPII budding (19,20) (Figure 1C,D).Both treatments block the loss of fluorescence from C1cells in the presence of AP21998, indicating that secretionhas been efficiently inhibited (no reporter construct wasdetected in the media of the brefeldin A experiment,data not shown). We have also determined the ability ofC1 cells, an adherent cell line, to secrete in suspension(Figure S1A) and the effect of inhibiting protein synthesison this assay (Figure S1B). Neither treatment significantlyaffected secretion of the reporter construct.

To determine if this system is amenable to ansiRNA-mediated approach, C1 cells were transfected withsiRNA against STX5, a SNARE previously shown to beessential for the ER-to-Golgi transport (21) and their abilityto secrete the reporter construct assayed. Immunoblot-ting of extracts generated from the C1 cells confirmed thatall three syntaxin 5 siRNAs efficiently depleted the shortand long isoforms of syntaxin 5 (Figure 2A, blot). Anal-ysis of the intracellular levels of the reporter constructby flow cytometry indicated that depletion of syntaxin 5blocked the loss of fluorescence from the cells in the pres-ence of AP21998 and the appearance of GH in the mediaas determined by immunoblotting (Figure 2A, graph andblot). These data suggest that syntaxin 5 is required forsecretion of the reporter construct. In confirmation of thisresult, it was observed that the transport of the reporterconstruct in syntaxin 5-depleted cells from the ER to Golgiwas drastically slowed compared with mock-transfectedcells as assayed by live-cell microscopy (Figure 2B andMovies S1 and S2).

SNARE and SM protein siRNA screen

To identify which SNAREs and SM proteins are requiredfor constitutive secretion, a Dharmacon OnTargetPlus

SMARTpool siRNA library targeting 38 SNAREs, 4SNARE-like proteins and 7 SM proteins was generated(see Materials and Methods: siRNA oligo sequences inSupporting Information) and transfected into C1 cells.The cells were grown for 72 h and then incubated withAP21998 for 80 min at 37◦C and their mean fluorescencedetermined using flow cytometry (Figure 3A). Under theseconditions, depletion of several SNAREs blocked secre-tion as determined by their fluorescent ratio being at leasttwice that of the mock-transfected cells (syntaxin 1B,syntaxin 5, syntaxin 8, syntaxin 18, SNAP-29, Sec22b,GS27, SLT1, Sec20 and the SM protein Sly1). To our sur-prise, siRNA targeted to VAMPs 3 and 8 (homologues ofSnc1p/2p), syntaxins 2, 3 and 4 (homologues of Sso1p/2p)and munc18-1, 2 and 3 (homologues of Sec1) failed toblock secretion. To determine whether the post-GolgiSNAREs were being effectively depleted using the 72-hknockdown protocol, cell extracts were generated fromthe cells and immunoblotted for VAMPs 3, 4, 7 and 8 aswell as the PM syntaxins 2, 3 and 4 (Figure S2A). Whilethe post-Golgi VAMPs were efficiently depleted usingthis protocol, the PM syntaxins were not. To resolve thistechnical problem, a double knockdown 96-h protocol (22)was tested and cell extracts generated from the cellsand immunoblotted for syntaxins 2, 3 and 4 (Figure S2C).The 96-h protocol significantly improved the depletions ofsyntaxins 2, 3 and 4, so the siRNA screen was repeatedusing this protocol (Figure 3B). The results obtained fromboth screens are consistent, with several additional genesbeing identified with the 96-h screen (syntaxin 17, syn-taxin 19 and Ykt6), indicating that incomplete depletion ofthe SNAREs was having an effect on the assay. Interest-ingly, depletion of syntaxin 1B and syntaxin 8 using the96-h-based protocol failed to produce a significant levelof inhibition, suggesting that the phenotype observed forboth genes may be off-target.

Validation of genes identified in the screen

With any siRNA screen it is important to confirm thatthe phenotypes observed are specific and not simplycaused by off-target effects (23). The best way of doingthis is by performing rescue experiments, where cellsare transfected with siRNA-resistant versions of thegene of interest. We have attempted to do this forSNAREs involved in the ER-to-Golgi transport. However,we have had very limited success, possibly because of thedominant-negative effects of SNARE overexpression (21).To overcome this problem, we have taken several over-lapping approaches. First, we have ascertained whichSNAREs are expressed in C1 cells by immunoblottingand microarray analysis (Figure S3A–C). Second, we havedeconvolved the siRNA SMARTpools (four per pool) andtested the oligos individually (Figure 4A). Third, we haveused immunoblotting to confirm the level of depletionwhen less than three out of the four siRNAs blocksecretion (Figure 4B). Fourth, we have determined wherethe reporter construct is blocked in the secretory pathway(Figure 5).

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Figure 2: Syntaxin 5 depletion using siRNA blocks secretion of the reporter construct. A) To determine if C1 cells were sensitiveto siRNA, C1 cells were either mock transfected (oligofectamine only) or transfected with three different syntaxin 5 siRNAs (100 nM)and grown for 72 h. The cells were then incubated with AP21998 (1 μM) for the indicated times at 37◦C and their mean fluorescencedetermined using flow cytometry (graph) and the appearance of reporter construct in the media determined by immunoblotting (60-mintime-point only). In addition, cell extracts were generated from these cells and immunoblotted to determine the degree of syntaxin 5depletion (blot). B) To determine where the block in secretion was occurring in the syntaxin 5-depleted cells, C1 cells were grown oncoverslips and either were mock transfected (oligofectamine only) or transfected with a syntaxin 5 siRNA-C (100 nM) and incubated for72 h. The cells were then incubated with AP21998 (1 μM) and imaged at 37◦C using live-cell confocal microscopy. Secretion was initiated30 seconds after recording began. Numbers indicate minutes. Scale bar: 50 μm. See Supporting Information for Movies S1 and S2. Insome cells the reporter construct can be found trapped/bound between the cell-cell contacts even in the absence of AP21998 (arrowheads).

The SNARE expression profiling indicated that most of thegenes identified in the siRNA screens were expressedin C1 cells (syntaxins 5, 8, 17, 18, GS27, SLT1, Sec20,Sec22b, SNAP-29 and Ykt6; Figure S3). We were unable todetect the expression of syntaxin 1B either by microarray-or immunoblotting-based approaches, suggesting that thephenotype observed in the syntaxin 1B knockdown ismost likely an off-target effect. We were also unable todetect the expression of syntaxin 19 using the microarraydata, although syntaxin 19 was represented on the chip.However, we were able to detect a syntaxin 19 transcriptusing Reverse transcription polymerase chain reaction

(RT-PCR) and that this transcript was depleted by treatingC1 cells with syntaxin 19 siRNA (Figure 4C).

To reduce the chances of falsely identifying genes ashaving a role in secretion because of off-target effects,we have deconvolved the siRNA SMARTpools andrescreened the individual siRNA in the secretion assay(Figure 4A). In the majority of cases, at least three out ofthe four siRNAs blocked secretion indicating that thesephenotypes are unlikely to be caused by off-target effects(syntaxin 18, syntaxin 19, GS27, SLT1, Ykt6 and Sly1). Onlyone out of the four siRNAs to syntaxin 1B and syntaxin

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Figure 3: A targeted siRNA screen to

determine which SNAREs and SM

proteins are required for secretion.

A) To determine which SNAREs andSM proteins are required for secre-tion of the reporter construct, C1 cellswere transfected with an OnTargetPlusSMARTpool siRNA library and grownfor 72 h. The cells were then incu-bated with AP21998 (1 μM) for 80 minat 37◦C and their mean fluorescencedetermined using flow cytometry. Dot-ted line represents a value twice thatof the mock-transfected cells. Error barrepresents the experimental range oftwo repeats. B) To determine whichSNAREs and SM proteins are requiredfor secretion of the reporter construct,C1 cells were transfected twice (time0 and 48 h) with an OnTargetPlusSMARTpool siRNA library and grownfor 96 h. The cells were then incu-bated with AP21998 (1 μM) for 80 minat 37◦C and their mean fluorescencedetermined using flow cytometry. Dot-ted line represents a value twice thatof the mock-transfected cells. Error barrepresents the experimental range oftwo repeats.

8 inhibited secretion, suggesting that the observed phe-notype is most likely an off-target effect. In some cases,only two out of the four siRNAs gave a phenotype sothese knockdowns were validated using immunoblotting(Figure 4B). For SNAP-29, syntaxin 17, Sec22B and Sec20,there was a reasonable correlation between the level ofknockdown detected by immunoblotting and the observedblock in secretion. In the case of the syntaxin 8 knock-down, there was no correlation between the level ofknockdown and the observed phenotype in the secretionassay, indicating that the block in secretion was causedby an off-target effect.

To determine where the reporter construct is accumulat-ing in the siRNA-depleted cells, we have performed fluo-rescence microscopy on C1 cells (Figure 5). C1 cells weretransfected with siRNA using the double knockdown pro-tocol and grown for 96 h and then incubated with AP21998for 80 min at 37◦C. The cells were fixed and then stainedfor GM130 (red), TGN46 (blue) and the reporter construct(GFP-green). In the mock-transfected cells, the majority ofthe reporter construct has been secreted from the cellsand is often found trapped/bound between the cell–cellcontacts. Depletion of syntaxin 18, Sec20 and Sly1 causes

the earliest block in secretion as the majority of thereporter construct has failed to leave the ER. In these cells,normal Golgi morphology is perturbed with TGN46 stainingalmost absent in the Sly1- and syntaxin 18-depleted cells.

Depletion of syntaxin 5, GS27, SLT1, Sec22b or Ykt6causes a later block in secretion as a significant amount ofthe reporter construct has been transported from the ERand has become trapped either in the Golgi or in very closeproximity to it. In addition, depletion of these SNAREs alsocauses Golgi fragmentation. In the Ykt6-depleted cells,there is also a significant accumulation of cargo in smallvesicles in the cytoplasm of the cell. To further character-ize this phenotype, electron microscopy was performedon Ykt6-depleted cells (Figure S4C). In the depleted cells,the Golgi is still stacked but is significantly swollen anddistended compared with mock-transfected cells. In addi-tion, there is a significant increase in the number ofelectron-lucent structures in close proximity to the Golgi.

Depletion of syntaxin 17, syntaxin 19 or SNAP-29 causesno apparent accumulation of reporter construct either inthe ER or Golgi. In addition, Golgi morphology in these

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Figure 4: Validation of library hits

with individual siRNAs. A) Tovalidate the genes identified in thescreen, C1 cells were transfectedwith individual oligos correspondingto those pooled in the libraryscreen using either the 72- or96-h-based protocols (* indicates96-h protocol). The cells were thenincubated with AP21998 (1 μM) for80 min at 37◦C and their meanfluorescence determined using flowcytometry. Error bar represents theexperimental range of two repeats.B) To determine the efficiencyof knockdowns, C1 cells weretransfected with individual oligoscorresponding to those pooled inthe library screen using the 72-h-based protocol and immunoblottingwas performed on cell extractsgenerated from these cells. C) Todetermine the efficiency of syntaxin19 knockdown, C1 cells were eithermock transfected (oligofectamineonly) or transfected with a syntaxin19 siRNA pool and harvested 48 hafter transfection for quantitativeRT-PCR.

cells appears to be relatively normal. A summary of thevalidation data is shown in Table 1.

Depletion of SNAP-29 and syntaxin 19 causes

a decrease in the number of fusion events at

the plasma membrane and in SNAP-29-depleted cells

there is an accumulation of docked secretory vesicles

at the cell surface

From what is known about the localization of syntaxin 19and SNAP-29, it is likely that they are involved in post-Golgitransport (24–26). To investigate this, C1 cells were eithermock transfected or transfected with siRNA to syntaxin 19and SNAP-29. Secretion of the reporter construct was initi-ated with AP21998 at 37◦C and the fusion of exocytic vesi-cles with the PM was measured using total internal reflec-tion fluorescence (TIRF) microscopy. Loss of syntaxin 19and SNAP-29 led to a significant decrease in the numberof fusion events at the cell surface with SNAP-29 siRNA-Acausing the most significant decrease (Figure 6A). Reas-suringly, SNAP-29 siRNA-A had previously been shown togive the strongest inhibition of secretion by flow cytome-try (Figure 4A) and cause the most complete depletion ofthe protein by immunoblotting (Figure 4B). In addition, wealso observed that depletion of SNAP-29 increased the

number of fluorescent structures/docked vesicles at thePM suggesting a block in fusion (Figure 6B,C).

Syntaxin 19 can form SNARE complexes with

SNAP-23, 25, 29 and VAMPs 3 and 8

To gain insight into the role of syntaxin 19 in constitutivesecretion, we have generated a stable cell line expressingan HA-tagged version of this protein and have analysedthe SNAREs it can interact with using mass spectrom-etry (MS). As previously published (24), the HA-taggedsyntaxin 19 construct is predominantly localized to thePM (Figure S5). HA immunoprecipitations performed onthese cells indicate that HA-tagged syntaxin 19 can inter-act with SNAPs 23, 25, 29 and VAMPs 3 and 8 (Table 2).No SNAREs were isolated in the control immunoprecipi-tations (data not shown).

Combinatorial knockdown of VAMPs 3, 4, 7 and 8

To our surprise, the siRNA screen failed to identify a post-Golgi R-SNARE required for the secretion of the reporterconstruct. In yeast, both the post-Golgi R-SNAREs Snc1pand Snc2p must be knocked out for a secretory phe-notype to be observed (27). If such redundancy existsin yeast, it is logical to assume that there will also be

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GFP (G)m

ock

ST

X5

ST

X17

ST

X18

ST

X19

Q-S

NA

RE

sR

-SN

AR

Es

SM

pro

tein

SN

AP

29G

S27

SLT

1S

ec20

Ykt

6S

ec22

BS

LY1

GM130 (R) TGN46 (B)

redundancy in mammalian cells. To test this hypothesis,we have performed a combinatorial knockdown wherewe have attempted to deplete all of the post-GolgiR-SNAREs expressed in the C1 cells as determined byimmunoblotting (Figure S3C). C1 cells were transfectedwith siRNA against VAMPs 3, 4, 7 and 8, as well as a com-bination of all four using the double knockdown protocol.The cells were then grown for 96 h and then incubatedwith AP21998 for 80 min at 37◦C and their mean fluores-cence determined using flow cytometry or the number ofexocytic fusion events quantified using TIRF microscopy.To determine the efficiency of knockdowns, cell extractswere generated from the cells and immunoblotted. A sig-nificant level of depletion was achieved for each SNAREindividually as well as in combination (Figure 7, blot).Despite this, no obvious block in secretion was detectedby flow cytometry (Figure 7, left graph) or TIRF microscopy(Figure 7, right graph).

Discussion

Development of a novel assay for measuring

constitutive secretion

There are many assays available for measuring constitu-tive secretion and they have played an important role indefining this pathway (28,29). The system we have devel-oped offers an alternate approach to these assays andovercomes some of their limitations. The reporter con-struct used in our assay is rapidly and efficiently secretedfrom cells with a half-time of secretion of approximately30 min, which is similar to that of other soluble pro-teins (30). The reporter construct is trafficked by the classi-cal secretory pathway as it is dependent on COPII vesicles,SNAREs and a functional Golgi. The assay does not requirechanges in temperature to initiate or maintain secretion ofthe reporter construct, thereby allowing for secretion to bemeasured at a physiological temperature. This assay is notaffected by changes in cell number as quantification of thesecretion is flow cytometry based. The assay is low cost,reproducible and amenable to high-throughput screeningprocedures, as secretion is directly measured without theneed for antibody staining. This approach also allows forsecretion to be measured using several different meth-ods such as flow cytometry, enzyme-linked immunosor-bent assay (ELISA), immunoblotting and fluorescentmicroscopy, allowing for the observed phenotypes to

Figure 5: Microscopy-based characterization of secretion

defects. To further characterize the defects observed insecretion, C1 cells were grown on coverslips and were eithermock transfected (oligofectamine only) or transfected twice (time0 and 48 h) with the indicated siRNA (100 nM) and incubated for96 h. The cells were then incubated with AP21998 (1 μM) for80 min at 37◦C, fixed and stained with anti-GM130 (red) andanti-TGN46 (blue). Scale bar: 10 μm. The images shown arerepresentative of the phenotype observed with two separatesiRNAs targeted to the same gene.

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Table 1: Summary of the validation data

SNARE orSM protein

72-hscreen

96-hscreen

Individual oligoswith phenotype

Western blot of KDcorrelates with data?

Cargo blocked inER/Golgi? Valid hit?

STX1B X — 1/4 Not expressed ND NoSTX5 X X 3/3 — Yes YesSTX8 X — 1/4 No ND NoSTX17 — X 2/4 Yes No YesSTX18 X X 3/4 — Yes YesSTX19 — X 3/4 — No YesSNAP-29 X X 2/4 Yes No YesYKT6 — X 4/4 — Yes YesSec22B X X 2/4 Yes Yes YesGS27 X X 3/4 — Yes YesSLT1 X X 3/4 — Yes YesSec20 X X 2/4 Yes Yes YesSly1 X X 4/4 — Yes Yes

ND, not determined.This table summarizes the validation and characterization data performed on the SNAREs initially identified in the screen.

be independently validated. The assay is also rela-tively robust in terms of sensitivity to off-target effectsas it allows for the levels of reporter construct tobe normalized for each knockdown. The aggregationsystem is also relatively flexible and can be usedto control the secretion of membrane-anchored pro-teins [A. Peden, unpublished observations – on thesecretion of vesicular stomatitis viral glycoprotein(VSV-G)-and glycosyl-phosphatidylinositol (GPI)-based con-structs]. Being able to assay the transport of solubleversus membrane-bound cargo will be useful in determin-ing if there are differences in the machinery required fortheir trafficking (31). We have also determined whetherthis system will work in other cell types (rodent andDrosophila cell lines) and preliminary experiments indicatethat these cells behave in a similar way to the HeLa-M(C1) cells (A. Peden, unpublished observations).

SNAREs in the ER-to-Golgi transport

The SNARE complexes consisting of syntaxin 5, Bet1,GS27, Sec22b or Ykt6 (anterograde) and syntaxin 18,SLT1, Sec20 and Sec22b (retrograde), two SNARE-likeproteins (Sec22A and C) and one SM protein (Sly1) havebeen proposed to play a role in transport between theER and Golgi in mammalian cells (11,32–36). All of thesegenes were expressed in HeLa-M (C1) cells as determinedby microarray analysis. Our siRNA screen has success-fully identified the majority of these SNAREs as well asthe SM protein Sly1, as being required for transport ofour synthetic cargo (Figure 8). Our findings indicate thatan siRNA-based approach can successfully be used tostudy the function of SNAREs. In addition to identifyingthe known SNAREs required for the ER-to-Golgi transport,we have detected a modest role for syntaxin 17 in thisprocess. Very little is known about the function of syntaxin17 other than that it is highly expressed in steroidogeniccells localized to smooth ER and is able to interact withSec22b, Bet1 and Sly1, supporting its role in anterogradetransport (37). It is possible that in more specialized cells

syntaxin 17 may be involved in the transport of a specificcargo. We have failed to detect any requirement for theSNARE Bet1 or the SNARE-like proteins Sec22A and Cin this process. However, because of a lack of antibodiesagainst these proteins, we have been unable to determinewhether we have successfully depleted them.

Unsurprisingly, knocking down the majority of theseSNAREs leads to a robust block in the ER-to-Golgi trans-port and also causes a significant perturbation of Golgimorphology. However, the elements of the Golgi are stillpresent in all of the knockdowns as defined by GM130staining (cis marker). This result is surprising if consideredin relation to the cisternal maturation model (38), as onewould predict that the Golgi should be consumed if thedelivery of new membrane and cargo by forward transporthas been inhibited. However, it may also be the case thatby blocking forward transport we are also stopping thedelivery of components of the machinery required for cis-ternal maturation and vesicle budding at the TGN leadingto a stabilization of the Golgi. Further investigation of therole of SNAREs in the ER-to-Golgi transport may providenovel insights into Golgi dynamics in mammalian cells.

SNAREs in Golgi to plasma membrane transport

Our siRNA screen has identified two post-GolgiQ-SNAREs, syntaxin 19 and SNAP-29, as being involvedin constitutive secretion. SNAP-29 (also known as GS32)is a homolog of the yeast SNARE Sec9p and has beenshown to bind both TGN and PM SNAREs in vivo (25,39).Biochemical and localization studies performed on NRKcells indicate that SNAP-29 is distributed between thecytosol and Golgi membranes (39). In neurons and oligo-dendrocytes, SNAP-29 is distributed between the cytosoland PM (40,41). In HeLa-M cells, we have been unableto localize endogenous SNAP-29. However, HA-taggedSNAP-29, in these cells, is cytosolic with some stainingat the PM (A. Peden, unpublished observation). Depletion

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Figure 6: Depletion of syntaxin 19 and SNAP-29 by siRNA

reduces the number of fusion events at the PM and in SNAP-

29-depleted cells causes an accumulation of vesicles under

the PM. A) To measure the number of vesicles fusing with thePM, C1 cells were grown on coverslips and either were mocktransfected (oligofectamine only) or transfected twice (time 0and 48 h) with SNAP-29 or syntaxin 19 siRNA (100 nM) andgrown for 96 h. The cells were then incubated with AP21998(1 μM) and imaged at 37◦C using TIRF microscopy. The datadisplayed represent the total number of fusion events in 10cells of each type, imaged over the period between 25 and60 min after the addition of AP21998. Error bars indicate standarddeviation. B) To measure the number of vesicles docked underthe PM, C1 cells were grown on coverslips and either were mocktransfected (oligofectamine only) or transfected twice (time 0and 48 h) with SNAP-29 or syntaxin 19 siRNA (100 nM) andgrown for 96 h. The cells were then incubated with AP21998(1 μM) and imaged at 37◦C using TIRF microscopy and thenumber of vesicles present in the evanescent field quantified[micrographs (a) mock, (b) SNAP-29 siRNA-A, (c) SNAP-29 siRNA-C, (d) syntaxin 19 siRNA]. The data shown in each column are asum of the number of vesicles visible in the evanescent field of10 cells. Error bars indicate standard deviation. Scale bar: 10 μm.

Table 2: Syntaxin 19 can form SNARE complexes with SNAPs23, 25, 29 and VAMPs 3 and 8

Protein Mascot scoreNumber of peptides

(unique) Coverage (%)

Syntaxin 19 505 32 (9) 28SNAP-23 415 19 (8) 44SNAP-25 280 6 (5) 25SNAP-29 274 14 (6) 23VAMP3 111 2 (2) 29VAMP8 85 3 (2) 12

To determine which SNAREs syntaxin 19 can interact withan anti-HA, immunoprecipitation was performed from cellseither expressing HA-tagged syntaxin 19 (Figure S5) or a non-transfected cell line as a negative control. The isolated complexeswere then analysed via mass spectrometry. No SNAREs weredetected in the negative control immunoprecipitation (data notshown). The table lists the SNAREs detected in the HA-taggedsyntaxin 19 immunoprecipitation.

of SNAP-29 in HeLa-M cells does not cause gross abnor-malities in Golgi morphology. However, this does not ruleout the possibility that SNAP-29 may function both at theGolgi and PM. Disruption of SNAP-29 function in humansleads to CEDNIK syndrome, a neurocutaneous syndromecharacterized by gross neuroanatomical defects and skinabnormalities (26). Transmission electron microscopy ofskin samples from patients with cerebral dysgenesis, neu-ropathy, ichthyosis, and keratoderma syndrome (CEDNIK)reveals abnormal lamellar granule formation and an accu-mulation of small vesicles in the cytoplasm, supportingSNAP-29’s role in secretion. In HeLa-M (C1) cells, thereare three additional SNAPs expressed (23,25,42) poten-tially explaining the partial block in secretion. However, forunknown reasons SNAP-29 appears to be preferentiallyused over these other SNAPs, possibly reflecting a prefer-ence for the SNAREs expressed in these cells. Very littleis known about the function of syntaxin 19 (also known assyntaxin 9). Tagged syntaxin 19 is localized to the cell sur-face and internal membranes (24). We have shown that itcan interact with the R-SNAREs VAMP3 and VAMP8 andthe Qbc SNAREs SNAP-23, 25 and 29, suggesting that itmay be acting as a Qa-SNARE. However, syntaxin 19 doesnot have a proteinaceous membrane anchor, potentiallyruling out a role in driving membrane fusion (43). It will beof interest to determine whether syntaxin 19 can mediatefusion in a liposome-based assay. Syntaxin 19 has beenshown to have a limited tissue distribution in mice, so itis unlikely that syntaxin 19 is the general Qa-SNARE forconstitutive secretion (24). Thus, we feel that syntaxin 19is more likely to have a role in the constitutive secretionof a specialized cargo. It will be of interest to determinesyntaxin 19’s distribution in human tissues.

We have failed to identify a post-Golgi R-SNARE requiredfor this process. There are seven post-Golgi R-SNAREsencoded in the human genome. Only VAMPs 3, 4, 7and 8 can be detected by immunoblotting in HeLa-M

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Figure 7: Combinatorial knockdown of post-Golgi R-SNAREs in C1 cells does not block secretion of the reporter construct.

To determine which post-Golgi R-SNAREs are required for constitutive secretion in C1 cells, C1 cells were transfected twice (time 0and 48 h) with siRNA to VAMPs 3, 4, 7 and 8 individually or as a pool and incubated for 96 h. The cells were then incubated withAP21998 (1 μM) at 37◦C for 80 min and their mean fluorescence determined using flow cytometry (left graph, error bar represents theexperimental range of three repeats) or the number of exocytic fusion events quantified using TIRF microscopy (right graph, error barsindicate standard error of the mean). The TIRF data represent the total number of fusion events in 10 cells of each type, imaged overthe period between 25 and 60 min after the addition of AP21998. To determine the efficiency of knockdowns, immunoblotting wasperformed on cell extracts generated from these cells.

Figure 8: Diagram indicating at what point on the secretory

pathway SNAREs isolated in the siRNA screen function.

Diagram showing a simplified secretory pathway in a non-specialized mammalian cell. Arrows indicate the direction oftransport.

cells. Depletion of these genes individually or in combi-nation failed to block secretion. In agreement with ourfindings, Okayama et al. (13) has shown that depletion ofVAMPs 3 and 8 individually or in combination failed to

block secretion, although these SNAREs were enrichedon secretory carriers. At present, it is unclear why we andothers have failed to block secretion by depleting post-Golgi R-SNAREs. It is possible that the residual levels ofthese SNAREs are still capable of driving fusion (44). Totest this hypothesis, we have investigated the effect ofknocking down R-SNAREs on the cell surface levels ofthe transferrin receptor (TF-R), as it has previously beenshown that VAMP3 is required for TF-R recycling (45).Depletion of VAMP7 did not have an effect, while lossof VAMP3 reduced the cell surface levels of the TF-Rby more than 60% (A. Peden, unpublished observations).This data suggests that the levels of knockdown we areobtaining are sufficient to inhibit post-Golgi transport. Sowhy are we unable to inhibit secretion? One possibility isthat another R-SNARE may be able to compensate for theloss of the post-Golgi R-SNAREs. Ykt6 is a good candidate,as it has been shown to be a promiscuous R-SNARE that isinvolved in many transport steps in yeast (46). At present,we are unable to test this hypothesis as depletion of Ykt6on its own completely blocks secretion (Figure 3B). It willbe of interest to determine if this inability to block secre-tion by knocking down post-Golgi R-SNAREs is also seenin other eukaryotic systems.

SM proteins in constitutive secretion

In agreement with the literature, we have found that theSM protein Sly1 is required for the ER-to-Golgi trans-port (47). Microarray analysis indicates that message forVps33A, Vps33B, Vps45, Munc18-1, 2 and 3 is present inHeLa-M cells. However, we have failed to identify a post-Golgi SM protein as being required for this process. As

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knocking down these proteins failed to give a phenotype,we have not investigated them further. It is possible thatour siRNA transfection protocol has failed to successfullydeplete these proteins. However, the simplest explanationfor why we failed to see a phenotype is redundancy. Workfrom Han et al.(52) has shown that Munc18-1 and 2 needto be depleted to obtain a complete block in regulatedsecretion. It will be of interest to perform a combinatorialknockdown of these proteins.

In conclusion, we have developed a novel flow cytometry-based assay for measuring constitutive secretion that issimple, robust and performed at physiological tempera-ture. We have shown for the first time that SNAP-29,syntaxin 17 and syntaxin 19 have a role in this process.This novel assay should allow for the rapid identificationand characterization of many new genes involved inconstitutive secretion.

Materials and Methods

Constructs and retroviral infectionsThe reporter construct used to generate the C1 cell line is based onthe pC4S1-FM4-FCS-hGH plasmid included in the RPD Regulated Secre-tion/Aggregation Kit (ARIAD Pharmaceuticals). Enhanced GFP (eGFP) (Clon-tech) was incorporated into the XbaI site according to company instructions(ARIAD Pharmaceuticals, 2002). The signal sequence (SS), eGFP, four FMaggregation domains, furin cleavage site (FCS) and human growth hormone(hGH) were then moved into the pQCXIP retroviral vector (Clontech) to gen-erate pQCXIP-S1-eGFP-FM4-FCS-hGH. This construct was then infectedinto HeLa-M cells as previously described (42). This virally transducedpopulation of cells was then autocloned using a MoFlow Flow cytometer(Beckman Coulter) based on GFP fluorescence. The clonal cell lines werethen screened for their ability to efficiently secrete the reporter construct.

HA-tagged syntaxin 19 was generated by PCR and cloned into the retroviralexpression vector pLXIN (Clontech). A stable population of cells expressingthis construct was generated as described earlier. Cyan fluorescent protein(CFP)-Sar1 (H79G) was a kind gift from D. Stephens.

Cell culture and transfectionsHeLa-M and Phoenix cells were grown in high glucose DME (PAA Lab-oratories) supplemented with 10% fetal calf serum, 50 IU/mL penicillin,50 μg/mL streptomycin and 2 mM glutamine (Sigma-Aldrich) at 37◦C ina 5% CO2 humidified incubator. Clone 1 cells were grown as abovewith the addition of 1.66 μg/mL puromycin. HeLa-M cells expressing HA-tagged syntaxin 19 were grown with the addition of 0.5 mg/mL G418(PAA Laboratories). Phenol red-free DMEM (PAA Laboratories) supple-mented as above with the addition of 50 mM HEPES buffer (Sigma-Aldrich)was used for time-lapse video microscopy experiments. CFP-tagged con-structs were transfected into C1 cells using HeLaMonster, according tothe manufacturer’s instructions (Mirus Bio LLC).

Antibodies and immunoblottingAntibodies were either generated in-house, purchased from commercialsuppliers or were gifts from other investigators. Please see SupportingInformation for a full list of antibodies used in this study. Sample preparationfor SDS–PAGE and immunoblotting has been previously described (48).

Flow cytometry-based secretion assaySecretion of the reporter construct was initiated using the small moleculeAP21998 (ARIAD Pharmaceuticals) at a concentration of 1 μM in prewarmed

culture media. To halt secretion, samples were placed on ice for 10 min,washed with cold PBS and trypsinized on ice for 2 h. To measure theamount of cargo remaining in the C1 cells after secretion, sampleswere analysed using an FACSCalibur flow cytometer fitted with a high-throughput sampler modification (BD Biosciences). When GFP and CFPneed to be analysed simultaneously, a Cyan-ADP flow cytometer (Beck-man Coulter) was used. Live cells were gated using forward and sidescatter as well as 7-aminoactinomycin-D (Invitrogen) exclusion. Between2000–10 000 live cells were analysed for each sample. The mean GFPfluorescence was calculated using the software FLOWJO (TreeStar). Forexperiments involving CFP-tagged proteins, CFP-expressing cells weregated prior to quantifying mean GFP fluorescence. The percentage of intra-cellular GFP remaining after secretion was calculated as a ratio between anunsecreted control well and an experimental well incubated with AP21998.

Immunofluorescence labelling and microscopyCell fixation and immunofluorescence labelling were performed as previ-ously described (42). Epifluorescence microscopy was performed using aZeiss Axioplan fluorescence microscope (Zeiss) equipped with a 63× oilobjective, a Hamamatsu Orca-R2 C10600 camera (Hamamatsu Photonics)and a SEDAT quad pass filter set (Chroma).

Time-lapse microscopy was performed at 37◦C using a Zeiss LSM510Meta microscope with a Plan-Apochromat 63×/1.4 oil digital interferencecontrast (DIC) lens. eGFP was excited using a 488-nm laser. In Movies S1and S2, an image was captured every 7 seconds and the movie is showing20 frames per second.

To measure vesicle fusion with the cell surface, cells were imaged using aZeiss Observer Z1 spinning disc and TIRF confocal microscope (Carl ZeissInc.). Images were acquired with a 100× lens, Hamamatsu PhotonicsEM-CCD digital camera (Hamamatsu Photonics) and AxioVision ImagingSoftware (Carl Zeiss Inc.). Cells were incubated at 37◦C during acquisition.During each TIRF experiment, a 488-nm argon laser was directed at thecoverslip at an angle greater than the critical angle of the laser line. Theresulting total internal reflection of the laser line created an evanescentfield in the 100–200 nm region of the sample closest to the coverslip. Sam-ples were imaged in 5-min intervals at maximum speed (approximately 30frames per second) at 25, 32, 39, 46 and 53 min after the addition of 1 μM

AP21998. Exactly two cells were imaged at each time-point to ensure thatthe distribution of cells over the course of imaging period was equivalentbetween mock and knockdown data sets. The number of vesicle fusionevents for each knockdown was determined by counting the number ofpunctate flashes of fluorescence in each movie. The total number of vesi-cles present at the base of a given cell was calculated for the first frameof each movie using Volocity Analysis software (Improvision).

siRNA transfectionsAn OnTargetPlus SMARTpool siRNA library targeting each individualSNARE in the human genome was purchased from Dharmacon.A complete list of siRNA target sequences can be found in SupportingInformation. Two siRNA transfection protocols were utilized in this study,both utilizing the transfection reagent oligofectamine (Invitrogen). The 72-hprotocol utilizes a single treatment with 100 nM siRNA at 0 h. The 96-hprotocol utilizes two treatments with 100 nM siRNA, one at 0 h and asecond at 48 h, followed by analysis at 96 h (22). To validate the genesidentified in the screen, the OnTargetPlus SMARTpools were deconvolvedand the four individual oligos tested.

Immunoprecipitation, trypsin digestion and peptide

analysisTo isolate HA-tagged syntaxin 19 SNARE complexes, immunoprecipita-tions were performed on detergent extracts generated from cell popula-tions pretreated with N-ethylmaleimide (100 μM) for 30 min at 37◦C. Thecells were resuspended in lysis buffer (100 mM NaCl, 5 mM MgCl2, 50 mM

Tris pH 7.4, 0.5% Igepal CA-630) with a complete protease inhibitor tablet

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(Roche Diagnostics Ltd), then cleared by centrifugation at 14462 × g andincubated for 2 h with anti-HA resin (Roche). Following multiple washsteps, the samples were eluted using 200 mM glycine at pH 2.3, thenacetone precipitated and stored at −80◦C. The samples were then trypsindigested using a filter-aided sample preparation (FASP) protocol (30-kDacutoff), as previously described (49). Peptides were concentrated usingC18 stage tips, made in-house from C18 material (3M) (50).

The tryptic peptides were analysed by reversed-phase LC-nanoESI-MS/MSusing a nanoAcquity (Waters) coupled to an LTQ-Orbitrap XL (ThermoFisher). Peptides were trapped and washed on a 180 μm × 20 mm C18Symmetry column (Waters) and resolved on a 75 μm × 150 mm BEH130C18 UPLC column (Waters). Peptides were eluted to the MS using a20-min 1–85% v/v Acetonitrile gradient flowing at 500 nL/min. Spectrawere acquired in positive mode with MS to MS/MS switching in a data-dependent manner. Each MS scan (resolution 60 000 fwhm at 400 m/z)was followed by five MS/MS scans of the most abundant molecular ionsfragmented by collision-induced dissociation using normalized collisionenergy of 35%. Spectra were processed using MAXQUANT version1.0.12.31 (51) and. msn files searched using Mascot Daemon 2.2.2 againstIPI human database release 20090422. Carbamidomethyl (C) was includedas a fixed modification and oxidation (M), N-acetylation (protein) anddeamidation (NQ) were included as potential variable modifications. Datawere searched with a peptide mass tolerance of 7 ppm and a fragmentmass tolerance of 0.5 Da. Peptides with an ion score below 28 (p < 0.05)were excluded. Proteins identified by single, unique peptide were manuallyvalidated and required a minimum of six consecutive b- or y-ions.

Quantitative RT-PCRRNA preparations were made using the QIAshredder Kit and the RNeasyProtect Kit (Qiagen). Combined reverse transcription and quantitative PCRwas performed using an RNA to CT kit (Applied Biosystems). Prestockedprobes against syntaxin 19 and the endogenous actin control were usedin the qRT-PCR reaction, and the products were run on a 3% agarose gelusing NuSieve GTG agarose (Cambrex).

Acknowledgments

The authors would like to thank Drs Matthew Seaman, Reinhard Jahn,Jesse Hay and Mitsuo Tagaya for generously sharing antibodies; DrDavid Stephens for the CFP-Sar1 construct and helpful advice; Drs RobinAntrobus, Michael Weekes and Georg Borner for their advice and assis-tance with proteomics and Dr Geoff Hesketh for his help with RT-PCR.We would also like to thank Drs Margaret Robinson and Sean Munrofor their helpful comments regarding this manuscript. We would alsolike to thank ARIAD Pharmaceuticals Inc. for providing the RegulatedSecretion/Aggregation Kit (http://www.ariad.com). Dr Peden is funded bya Career Development Award from the Medical Research Council.

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1: Characterization of C1 secretion kinetics in suspension

and the influence of protein synthesis on the secretion assay. A) Todetermine the ability of C1 cells to secrete in suspension, C1 cells weretrypsinized and resuspended in DMEM supplemented with HEPES buffer.The C1 cells were then incubated with or without (1 μM) AP21998 forthe indicated times at 37◦C while being agitated. In parallel, a secretionexperiment was performed with adherent C1 cells. The mean fluorescenceof both sets of samples was determined by flow cytometry. B) Todetermine if protein synthesis affects the secretion assay, C1 cells wereincubated either in the presence or absence of cyclohexamide (100 μg/mL)and AP21998 (1 μM) for the indicated times at 37◦C and their meanfluorescence determined using flow cytometry.

Figure S2: Analysis of post-Golgi SNARE depletion using different

knockdown protocols. A) To determine the efficiency of post-GolgiSNARE depletion using a 72-h-based protocol, C1 cells were transfectedwith siRNA targeting VAMPs 3, 4, 7 and 8 and syntaxins 2, 3 and 4 (siRNApools are identical to those used in the secretion screen) and incubatedfor 72 h. The cells were then harvested and immunoblotted for VAMPs3, 4, 7 and 8 and syntaxins 2, 3 and 4. B) To determine the efficiency ofpost-Golgi Q-SNARE depletion using a 96-h- based protocol, C1 cells weretransfected with siRNA twice against syntaxins 2, 3 and 4 and incubatedfor 96 h. The cells were harvested, detergent extracts generated fromthem and normalized for protein expression using the Bradford assay andthen resolved by SDS–PAGE and immunoblotted for syntaxins 2, 3 and 4.

Figure S3: Analysis of SNARE and SM protein expression in HeLa-

M cells using both microarray and immunoblotting. A) Tablesummarizing microarray and immunoblotting data for SNARE and SMprotein expression. B) To determine which SNAREs are expressed inHeLa-M cells, detergent extracts were generated from HeLa-M cells andcontrol samples’ HeLa, mouse brain, heart and cytotoxic T cells. Theextracts were then normalized for protein expression using the Bradfordassay and then resolved by SDS–PAGE and immunoblotted with theindicated antibodies. Numbers indicate molecular weight in kDa. C) Todetermine which SNAREs are expressed in HeLa-M cells, RNA wasextracted from HeLa and HeLa-M cells in duplicate and analysed by theUniversity of Cambridge Microarray Resource Center. The data obtainedwere analysed using the lumi R package and the resulting signal intensityvalues are close to a log2 transformation. Probes with signal intensityabove the background in at least one of the technical replicates of either ofthe cell lines were selected using a p-value threshold of <0.01 (performedby Julien Bauer, University of Cambridge Microarray Resources Centre).Approximately 11 008 genes were determined as being expressed in theHeLa-M cells. The table shows the SNAREs and SM proteins that weredetermined as being expressed.

Figure S4: Depletion of YKT6 affects Golgi morphology. A) Tocharacterize the YKT6 antibody and confirm the ability of the YKT6 siRNA toefficiently deplete YKT6, HeLa-M cells were grown on coverslips and wereeither mock transfected (oligofectamine alone) or transfected twice (time0 and 48 h) with an OnTargetPlus SMARTpool against YKT6 and incubatedfor 96 h. The cells were then fixed and stained with anti-YKT6 antibodies.Scale bar: 20 μm. B) To determine where YKT6 is localized in mammaliancells, HeLa-M cells were fixed and stained with anti-YKT6, anti-GM130and anti-TGN46 antibodies. Scale bar: 20 μm. C) To determine what effectYKT6 depletion has on Golgi morphology, HeLa-M cells were either mocktransfected (oligofectamine alone) or transfected twice (time 0 and 48 h)with an OnTargetPlus SMART pool against YKT6 and incubated for 96 h.The cells were then fixed and processed for EM. Mock cells: panels(a) and (c), YKT6 knockdown cells: panels (b) and (d), (G) Golgi complex,(PM) plasma membrane, (E) endosome. Arrows indicate budding profiles.Scale bar: 500 nm.

Figure S5: Localization of HA-STX19. To determine where STX19 islocalized within the cell and to identify which SNAREs STX19 interactwith, HA-STX19 was retrovirally transduced into HeLa-M cells and a stablepopulation of cells selected. Non-transduced HeLa-M cells (A) and HA-STX19-expressing HeLa-M cells (B) were stained with anti-HA antibodies.Scale bar: 20 μm.

Movie S1: C1 cells were grown on coverslips and incubated withAP21998 (1 μM) and imaged at 37◦C using live-cell confocal microscopy.Secretion was initiated 30 seconds after recording began. An image wascaptured every 7 seconds and the movie is showing 20 frames per second.

Movie S2: C1 cells were grown on coverslips and were transfectedwith an STX5 siRNA (100 nM) and incubated for 72 h. The cells were thenincubated with AP21998 (1 μM) and imaged at 37◦C using live-cell confocalmicroscopy. Secretion was initiated 30 seconds after recording began. Animage was captured every 7 seconds and the movie is showing 20 framesper second.

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Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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