JCS014225 Supplementary Material

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• Supplemental Table S1 -

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• Supplemental Figure S1 (Adobe PDF) -

  Fig. S1. Dominant-negative Rab43 causes Golgi fragmentation. (A) HeLa cells expressing GFP-Rab43 wild type, the Q77L activated mutant, or the T32N dominant-negative mutant (all green) were stained for the Golgi marker GM130 (red). (B) HeLa cells expressing GFP-Rab43 T32N (green) were stained for the markers indicated in the figure (red). DNA was stained with DAPI. Bar, 10 μm, in all panels.

• Supplemental Figure S2 (Adobe PDF) -

  Fig. S2. Dominant-negative Rab43 perturbs retrograde but not anterograde transport to the Golgi. (A) VSV G transport assays were performed in HeLa cells expressing Myc-tagged dominant-negative Rab43 T32N or RN-tre (blue). After 60 minutes of transport cells were fixed and surface stained for VSV G (red), then permeabilized and stained for the Myc epitope (blue). Total VSV G was observed by GFP fluorescence (green). (B) Shiga toxin (STxB) transport assays were performed in HeLa cells expressing GFP-tagged dominant-negative Rab43 T32N or RN-tre (green). After 60 minutes of transport cells were fixed and permeabilised, then stained for the Golgi marker GM130 (blue). STxB-Cy3 conjugate was visualized directly (red). DNA was stained with DAPI in RN-tre-expressing cells (blue). Bar, 10 μm, in all panels.

• Supplemental Figure S3 (Adobe PDF) -

  Fig. S3. TBC1D20 is a highly conserved Rab GAP. TBC1D20 sequences identified in human (hs), macaque (ma), mouse (mm), rat (rn), dog (cf), cow (bt), zebrafish (dr), African clawed toad (xl), and three yeasts: Saccharomyces cerevisiae (scGyp8), Schizosaccharomyces pombe (spGyp10), and Kluveromyces lactis (klTBC1D20) were aligned using ClustalX. Partial sequences could be found in other species, for example chicken, but these were omitted for simplicity. Identical amino acids are coloured red on a yellow background, high conservation is shown in dark blue on a light blue background, and similarities by dark blue on a green background. Red arrowheads mark the conserved catalytic argentine and glutamine residues. A conserved hydrophobic region close to the C-terminus predicted to form a transmembrane domain is underlined in green.

• Supplemental Figure S4 (Adobe PDF) -

  Fig. S4. Depletion of Rab2 disrupts Golgi structure but not secretion. (A) Cell lysates from HeLa cells expressing GFP-Rab2 treated with control or Rab2-specific siRNA duplexes were western blotted for GFP and α-tubulin as a loading control. (B,C) HeLa cells expressing GFP-Rab2 (green) were treated with control or Rab2-specific siRNA duplexes for 72 hours were fixed, and then stained with antibodies to the Golgi marker GM130, or the Sec31 subunit of the COPII vesicle coat (red). (D) VSV G transport assays were performed in HeLa cells treated for 72 hours with control or Rab2-specific siRNA duplexes. After 60 minutes of transport, cells were fixed and surface stained for VSV G (red). Total VSV G was observed by GFP fluorescence (green). DNA was stained with DAPI (blue). Bars, 10 μm.

• Supplemental Figure S5 (Adobe PDF) -

  Fig. S5. COPII clustering in the Golgi region requires the p115-GM130 tether. (A) Cell lysates from HeLa cells treated with control, p115 or GM130-specific siRNA duplexes were western blotted for GM130 or p115, and α-tubulin as a loading control. (B) HeLa cells treated with control, p115, or GM130 siRNA duplexes for 72 hours were fixed, and then stained with antibodies to p115, GM130, GRASP55, and COPII as indicated. The antibodies in the leftmost column are green, and those in the centre column are red in the merged image shown to the right. DNA was stained with DAPI (blue). Bars, 10 μm.