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First published online 8 March 2005
doi: 10.1242/jcs.01710

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O-glycosylation is essential for intracellular targeting of synaptotagmins I and II in non-neuronal specialized secretory cells

¹ Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel
² Fukuda Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan

View larger version (13K): [in a new window]   Fig. 1. Schematic representation of constructs used in this study. The diagrams show the positions of the chimeric proteins comprising Syt I and Syt II. C, cytosolic domain; HA, hemagglutinin; J, juxtamembrane domain; L, luminal domain; T7, 11 residue peptide (MASMTGGQQMG) derived from the major capsid protein of the T7 phage; TM, transmembrane domain.

View larger version (34K): [in a new window]   Fig. 2. Cellular localization of Syt I and Syt II. (A) RBL cells transiently transfected with T7-Syt I, HA-Syt II or stably transfected with Syt II were grown on glass coverslips for 24 hours. Cells were subsequently labeled with anti-T7 monoclonal antibodies (a), anti-HA monoclonal antibodies (b) or anti-C2A-Syt IX polyclonal antibodies (c) followed by Cy3-conjugated donkey anti-mouse or Rhodamine-conjugated donkey anti-rabbit IgG. Cells were processed for immunofluorescent staining and visualized by confocal microscopy. (B) Subcellular fractionation of T7-Syt I or HA-Syt II-transfected RBL cells. Cell homogenates derived from RBL cells transfected with either T7-Syt I or HA-Syt II cDNA were fractionated on continuous sucrose gradients as described previously (Baram et al., 1999). Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-T7, anti-HA or anti-Gi2 antibodies. Fractions were also examined for ß-hexosaminidase activity (presented as OD at 405 nm). Bar, 10 µm.

View larger version (75K): [in a new window]   Fig. 3. Visualization of the cellular localization of Syt I and Syt II chimeric proteins. (A) RBL cells transiently double transfected with HA-Syt I/Syt II and T7-Syt I cDNA were grown on glass coverslips and labeled with polyclonal anti-HA and monoclonal anti-T7 antibodies followed by Rhodamine-conjugated donkey anti-mouse and FITC-conjugated donkey anti-rabbit IgG. (B) RBL cells transiently transfected with HA-Syt I/Syt II cDNA were grown on glass coverslips and double stained with polyclonal anti-HA and monoclonal anti-serotonin antibodies followed by Rhodamine-conjugated donkey anti-rabbit and FITC-conjugated donkey anti-mouse IgG. (C) RBL cells, transiently transfected with HA-Syt II (1-183aa)/Syt I (a), HA-Syt II (1-86aa)/Syt I (b) or HA-Syt II (1-60aa)/Syt I (c) cDNA, were grown on glass coverslips and stained with monoclonal anti-HA antibodies. Cells were processed for immunofluorescent staining and visualized by confocal microscopy. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 4. Subcellular fractionation of HA-Syt I/Syt II- or HA-Syt II/Syt I-expressing RBL cells. Cell homogenates derived from RBL cells transiently transfected with HA-Syt I/Syt II or HA-Syt II(1-183aa)/Syt I cDNA were fractionated on continuous sucrose gradients as described previously (Baram et al., 1999). Fractions were collected from the top, subjected to SDS-PAGE and immunoblotted with anti-HA antibodies. Fractions were also examined for ß-hexosaminidase activity (presented as OD at 405 nm).

View larger version (63K): [in a new window]   Fig. 5. Biosynthetic routes of Syt I and Syt II. RBL cells, transiently transfected with Syt II (a-c) or T7-Syt I (d-g) cDNA, were grown on glass coverslips for 1 hour (a-f) or 5 hours (g) and then incubated for 2 hours at 20°C (a-f) or for 1 hour at 4°C in the presence of monoclonal antibodies directed against T7 (1 µg/ml) (g). Cells were subsequently transferred to 37°C (time zero) and incubated for the indicated time periods. Cells were stained with polyclonal antibodies directed against C2A-Syt IX (a-c) or with monoclonal antibodies directed against T7 (d-f) followed by Cy3-conjugated secondary antibodies. Cells were visualized by laser confocal microscopy. Bar, 5 µm.

View larger version (71K): [in a new window]   Fig. 6. Effect of inhibitors of internalization on HA-Syt I/Syt II trafficking. RBL cells transiently transfected with HA-Syt I/Syt II cDNA were grown on glass coverslips for 1 hour and subsequently incubated for 2 hours at 20°C (a). Cells were then warmed to 37°C for 3 hours in the absence (b) or presence of chloroquine (100 µM) (c) or sucrose (0.45 M) (d). Cells were subsequently stained with monoclonal anti-HA antibodies followed by Cy3-conjugated secondary antibody. Cells were processed for immunofluorescence and visualized by laser confocal microscopy. Bar, 5 µm.

View larger version (91K): [in a new window]   Fig. 7. Cellular localization of GFP-tagged Syt II. RBL cells stably transfected with Syt II-GFP (A) or Flag-Syt II-GFP (A,B) were grown on glass coverslips and either left untreated (A) or were serum starved for 1 hour and subsequently incubated for the indicated time periods with Texas Red-conjugated Tfn (50 µg/ml) (B). Cells were fixed and visualized by laser confocal microscopy. Bar, 10 µm.

View larger version (48K): [in a new window]   Fig. 8. Western blot analysis of HA-Syt II expressed in RBL cells. Cell extracts (80 µg) derived from RBL cells stably transfected with either (A) non-tagged Syt II (1) or HA-Syt II (2) cDNA, or (B) Syt II-GFP or Flag-Syt II-GFP cDNA or (C) transiently transfected with T7-Syt I, T7-Syt I(T15/16/A) or T7-Syt I(T26/A) cDNA, were resolved by SDS-PAGE and subjected to immunoblotting with either monoclonal anti-Syt II or anti-HA antibodies as indicated (A), or anti-GFP antibodies (B), or anti-T7 antibodies (C). The positions of the molecular mass markers are shown on the right in A. Arrows point to fully, partially or non-modified immunoreactive proteins.

View larger version (29K): [in a new window]   Fig. 9. Analysis of glycosylation of tagged Syt I and II. (A) RBL cells transiently transfected with T7-Syt I, HA-Syt II or the chimeras HA-Syt I/Syt II and HA-Syt II(1-183aa)/Syt I cDNA were grown for 2 hours at 37°C and subsequently left untreated or incubated for 24 hours in the presence of tunicamycin (10 µg/ml). Cell lysates were resolved by SDS-PAGE and probed with either anti-T7 or anti-HA antibodies, as indicated. (B) Cell lysates derived from RBL cells transfected with T7-Syt I, HA-Syt II, HA-Syt I/Syt II and HA-Syt II(1-183aa)/Syt I cDNA were treated with N-glycosidase F or buffer as described in Materials and Methods. Lysates were subjected to SDS-PAGE and probed with anti-T7 or anti-HA antibodies. (C) Cell lysates (80 µg) derived from RBL cells stably transfected with Syt II-GFP or Flag-Syt II-GFP were treated with N-glycosidase F, resolved on SDS-PAGE and probed with anti-GFP antibodies. The asterisks in panels A-C indicate proteins that were reduced by tunicamycin or N-glycosidase F treatment. (D) RBL cells transfected with Syt II(T34/A)-GFP or Flag-Syt II(T34/A)-GFP were subjected to tunicamycin or (E) N-glycosidase F treatment as described above. (F) Lysates (80 µg) derived from RBL cells transiently transfected with Flag-Syt II-GFP (WT)(1, 5) or Flag-Syt II(T34/A)-GFP (N)(2), or Syt II-GFP (WT)(3) or Syt II(T34/A)-GFP (N)(4) or Flag-Syt II(T17/18/19/A)-GFP (O) (6) were subjected to SDS-PAGE and probed with anti-GFP antibodies. Lane 3 is a longer ECL exposure time of the lane marked 3*.

View larger version (44K): [in a new window]   Fig. 10. Visualization of T7-Syt I and Syt II glycosylation mutants. RBL cells transiently transfected with T7-Syt I(T26/A) (a) or T7-Syt I(T15/16/A) (b) or Syt II(T17/18/19/A) (c) were grown on glass coverslips for 24 hours and subsequently labeled with anti-T7 monoclonal antibodies (a,b) or polyclonal antibodies directed against C2A-Syt IX (c) followed by the relevant Cy3-conjugated (a,c) or Cy2-conjugated secondary antibodies. Cells were processed for immunofluorescent staining and visualized by confocal microscopy. The z-line image is of the same field as in b and confirms the plasma membrane localization of the O mutant. Bar, 10 µm.