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First published online 27 March 2007
doi: 10.1242/jcs.03436

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Syntaxin 16 and syntaxin 5 are required for efficient retrograde transport of several exogenous and endogenous cargo proteins

Mohamed Amessou^1,*, Alexandre Fradagrada^1,*, Thomas Falguières¹, J. Michael Lord², Daniel C. Smith², Lynne M. Roberts², Christophe Lamaze¹ and Ludger Johannes^1,

¹ Traffic and Signaling Laboratory, UMR144Curie/CNRS, Institut Curie, 26 rue d'Ulm, F-75248 Paris Cedex 05, France
² Molecular Cell Biology Group, Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK

View larger version (60K): [in this window] [in a new window]   Fig. 1. Specific inhibition of retrograde transport of STxB in Syn16 RNAi conditions. (A) Metabolic sulfation of internalized STxB-Sulf2 on HeLa cells over a 20-minute period was visualized by immunoprecipitation from cells that had been transfected for 72 hours with three different siRNAs against Syn16 or with scrambled (Sc) siRNA (200 nM each). Data are expressed as percentages of STxB-Sulf2 sulfation for each experimental condition, using sulfation of STxB-Sulf2 on mock-transfected cells as the reference (set to 100%), and normalized for total sulfation counts (see Materials and Methods and Fig. 2C). Inset: Western blotting confirmed the down-modulation of Syn16 under these conditions. Tubulin was used as a loading control. (B) Sulfation analysis (as in A) of STxB-Sulf2 applied to HeLa cells that had been transfected for 72 hours with increasing doses of anti-Syn16 siRNA number 3. Inset: Western blot of Syn16 under the indicated conditions. (C-E) Immunofluorescence analysis of retrograde STxB transport in (C) mock-transfected control (CTL) and (D-E) Syn16 RNAi cells (siRNA sequence number 3 at 200 nM). In all conditions fluorophore-labeled STxB (green) was internalized for 45 minutes at 37°C. Cells were fixed and labeled for Syn16 (blue), the Golgi marker CTR433 (red, D), or TfR (red, E). Under Syn16 RNAi conditions, STxB accumulation in the Golgi was reduced, and the protein appeared in peripheral structures where it co-distributed with the TfR. Bar, 10 µm. (F-H) Specificity controls for the Syn16 RNAi. (F) EGF degradation. 2.5 ng/ml of iodine-labeled EGF was incubated for the indicated times with HeLa cells in the indicated conditions. Lysosomal degradation was measured by appearance of TCA-soluble counts in the culture medium (G) Tf recycling. Biotin-tagged Tf was internalized at 37°C for 40 minutes into HeLa cells in the indicated conditions. Cells were washed and further incubated as shown at 37°C. Remaining cell-associated Tf was determined by ELISA. In F-G, the means of three to four independent experiments are shown. (H) VSVG transport in the biosynthetics/secretory pathway. The trafficking of newly synthesized VSVG-ts045 to the plasma membrane was analyzed by cell-surface biotinylation after a shift in temperature from 40°C to 32°C. BFA was used as a specificity control.

View larger version (72K): [in this window] [in a new window]   Fig. 2. Effect of Syn5 RNAi on cell entry by STxB. (A) Western blot analysis of target-protein expression under variable RNAi conditions. Note that, for Syn5, two different isoforms of the protein were detected. Sc,scrambled. (B) Sulfation was monitored over 20 minutes following addition of STxB-Sulf2 to HeLa cells that had been transfected for 72 hours with 200 nM scrambled siRNA or siRNAs targeting the indicated proteins. For Syn5, two independent siRNA sequences were used that gave similar results. For Syn16, sequence number 3 was used. Data are expressed as percentages of STxB-Sulf2 sulfation for each experimental condition, using sulfation of STxB-Sulf2 on mock-transfected cells as the reference (set to 100%), and normalized for total sulfation counts (see C). RNAi against the late endosomal Syn7 was taken as a specificity control. Mean ± s.e.m. of three to five determinations are shown. (C) Sulfation levels on endogenous proteins under the indicated experimental conditions. (D-F) Immunofluorescence analysis of retrograde STxB transport in (D) mock-transfected control (CTL) or (E-F) Syn5 RNAi cells. In all conditions, fluorophore-labeled STxB (green) was internalized for 45 minutes at 37°C. Cells were fixed and labeled for Syn5 (blue), the (D-E) Golgi marker CTR433 (red), or (F) TfR (red). Under Syn5 RNAi conditions, STxB accumulation in the Golgi was reduced, and the protein appeared in peripheral structures where it co-distributed with TfR. Note that Golgi membranes were partly dispersed in (E) Syn5 RNAi cells, CTR433). Bar, 10 µm.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Analysis of endocytosis and TGN/Golgi-to-ER transport in Syn5 RNAi cells. (A) Glycosylation analysis. Iodinated STxB-Glyc-KDEL was incubated with mock-transfected or RNAi-transfected HeLa cells for 4 hours. The percentage of glycosylated STxB-Glyc-KDEL was determined from gels, as described (Johannes et al., 1997; Mallard and Johannes, 2003). Glycosylation of STxB-Glyc-KDEL in mock-transfected cells was taken as the maximal possible (100%) value. Mean ± s.e.m. of two to three experiments. (B,C) The endocytosis of (B) STxB and (C) Tf was determined on the same cells. Data represent the percentage of cell-surface inaccessible biotin-tagged STxB or Tf at each time point. Mean ± s.e.m. of four experiments. (D,E) Quantitative analysis of TGN/Golgi-to-ER transport. (D) STxB-Sulf-Glyc-KDEL was internalized into mock-transfected (± tunicamycin) or siRNA-transfected HeLa cells for 2 hours in the presence of radioactive sulfate and then chased for 4 hours at 37°C. Cells were lysed, STxB-Sulf-Glyc-KDEL immunoprecipitated and analyzed by SDS-PAGE. The uppermost band (1) corresponds to the glycosylation product which disappears upon tunicamycin treatment. See text for details. (E) Quantitative analysis by Phosphoimager of the mean of two to three experiments ± s.e.m., as shown in D. Data express the percentages of sulfated STxB-Sulf-Glyc-KDEL that also become glycosylated in each experimental condition.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Retrograde transport of different cargo proteins in Syn5 RNAi and Syn16 RNAi cells. (A) Constructs used for sulfation analysis. A genetic fusion of STxB with tandem sulfation sites was made as described (Mallard et al., 1998). For CTxB, a tandem-sulfation-site peptide was chemically coupled to primary amines. In the case of MPR, the tandem-sulfation-site peptide was chemically coupled to an anti-GFP antibody recognizing MPR-GFP protein in stably transfected HeLa cells (Waguri et al., 2003). (B) Sulfation analysis of sulfation-site -peptide-coupled CTxB and MPR. HeLa cells were incubated for 40 minutes with the indicated proteins in the presence of radioactive sulfate with or without BFA, followed by immunoprecipitation and autoradiography. For CTxB, the arrow indicates sulfated toxin-B subunit, whereas the upper bands, visible under conditions with or without BFA, originate from contaminating endogenous sulfoproteins at the level of the load. (C,D) STxB-Sulf2, CTxB-Sulf2 and sulfation-site-coupled anti-GFP-MPR antibody were incubated for 40 minutes in the presence of radioactive sulfate with HeLa cells that had been transfected with (C) Syn16 siRNA number 3 or (D) Syn5 siRNA number 1. Sulfated proteins were immunoprecipitated and analyzed by SDS-PAGE and autoradiography. Sulfation signals are expressed as normalized percentages of sulfation observed in mock-transfected control cells.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Co-distribution analysis of STxB and CTxB. (A,B) Immunofluorescence analysis of STxB (red) and CTxB (green) distribution in (A) Syn16 RNAi or (B) Syn5 RNAi cells after internalization for 45 minutes at 37°C. (C) Competition analysis. Iodinated STxB was incubated with HeLa cells in the presence of increasing concentrations of non-labeled CTxB or STxB. The amount of cell-associated radioactivity under each condition was determined. Note that CTxB did not displace the iodinated STxB from its receptor. (D) Binding analysis. HeLa cells were incubated on ice with 1 µM Alexa Fluor-488-labeled CTxB and 1 µM Cy3-labeled STxB. The cells were washed and fixed, and numbers of cells positive for one or the other marker were counted. Double-positive cells are roughly present at numbers as expected if GM1 and Gb3 expressions are independent events.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Syn5 and Syn16 RNAi effects on cell intoxication by STx, CTx and ricin. (A) Mock-transfected control cells or HeLa cells transfected with the indicated siRNAs were treated for 1 hour at 37°C with the indicated doses of STx. Protein biosynthesis was then determined by measuring the incorporation of radiolabeled methionine into acid-precipitable material. Data are presented as percentage of protein biosynthesis under the indicated conditions when compared with non-toxin-treated cells. (B) Cells were treated for 1 hour with the indicated doses of STx and analyzed as described in A. (C) Mock-transfected or RNAi-transfetced cells were treated with increasing doses of CTx, and cAMP production was measured to detect CTx arrival in the cytosol. (D) Mock-transfected or RNAi-transfetced cells were treated for 1 hour with the indicated doses of ricin, and analyzed as described in A. (E) Intoxication of HeLa cells by un-nicked (STx) or pre-nicked STx (Nicked) in mock-transfected control cells or Syn5 RNAi cells (siSyn5). Pre-nicking did not alter the Syn5-RNAi-mediated protection of cells against the toxin. (A-E) Means of two to four experiments.