JCS01314 Supplemental Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Punctate Ykt6 staining is independent of tagging and fixation methods. (A) HA-tagged yeast Ykt6p constructs were expressed in PC12 cells. Subcellular localization of HA-tagged full-length yeast Ykt6p (top left) and yeast Ykt6p-lgn domain (bottom left) was examined by double-label fluorescent microscopy using anti-HA antibody (16B12) and anti-rat-Ykt6 antibody. Arrows point to punctate structures where the HA-tagged proteins and endogenous rat Ykt6 colocalize (overlap was 18% and 17%, respectively). Scale bar, 10 mm. (B) Myc-tagged full-length Ykt6p (top left) and Myc-tagged Ykt6p mutants (bottom row) were expressed in PC12 cells. Cells were fixed with methanol and the subcellular localization of each recombinant protein and endogenous rat Ykt6 was examined by double-label fluorescence microscopy using anti-Myc antibody (9E10) and anti-rat-Ykt6 antibody. Arrows point to punctate structures where the Myc-Ykt6p and endogenous rat Ykt6 colocalize (top row; 24% overlap). For F39E, V8N and E100K/Y101N, only the subcellular localization of Myc-tagged mutant proteins is shown (bottom row). Scale bar, 10 mm.

• Supplemental Figure 2 -

  Fig. S2. (A) Subcellular localization of lipidated and non-lipidated SNARE motif. PC12 cells were transfected with expression constructs encoding Myc-rYkt6 137-198 or Myc-rYkt6 137-193. To examine the subcellular localization of each SNARE motif, cells were fixed at 24 hours after transfection and stained with anti-Myc antibody (9E10). (B) Results of bimolecular binding assay performed in octyl-glucopyranoside. Assay protocols and material preparations are essentially identical to Fig. 8. After transfecting PC12 cells with Myc-rYkt6 137-198, ligand-containing fraction was prepared in lysis buffer containing 2% (w/v) octyl-glucopyranoside. The bimolecular binding assay was performed in the presence of 0.285% octyl-glucopyranoside [the CMC of this detergent is ~0.7% (w/v)]. Arrow points to the protein band that corresponds to Myc-SNARE motifs.