Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1 - The tER-Golgi labelling is greatly reduced upon dGRASP depletion by RNAi.
  Mock- (A) and dGRASP/dGM130 depleted cells (B) for 96h were labelled with the anti-GRASP65 antibody. Note that, in contrast to the mock-depleted cells, the tER-Golgi area (in brackets) in the depleted cells is devoid of labeling (the overall labeling density is reduced by 5). ER, endoplasmic reticulum; PM, plasma membrane. Bars: 200nm.
• Supplemental Figure 2 - Detection of dGRASP-GFP chimeras by Western blotting
  Extracts from 0.5 million S2 cells transiently transfected with full length dGRASP-GFP, N-ter dGRASP-GFP, C-ter dGRASP-GFP, G2A-dGRASP-GFP, empty GFP vector, or without any vector were processed for Western blotting using an anti-GFP (A) or the anti-GRASP65 antibody (B). Most of the full length dGRASP-GFP appears to be dimerised (band 2), while only a small portion is detected in its monomeric form running at ~80kD (band 1). Bands 3, 4 and 5 correspond to the expected molecular weight of N-ter, C-ter and G2A dGRASP-GFP chimeras, respectively. Band 6 is likely to represent a degradation product of G2A mutant. Note that all bands are recognised by the anti-GRASP65 antibody with the exception of the C-ter-GFP mutant protein. In blot B, the arrow indicates the endogenous monomeric dGRASP.
• Supplemental Figure 3 - Effects of BFA and H89 on the organisation of the exocytic pathway in S2 cells
  Cells incubated without (A-C), with 50μM BFA for 30 minutes at 37°C (D-F), and with 50μM H89 for 2h at 27°C (G-I) were labelled for dSec23p (A,D,G, green) and d120kd (B,E,H, red). Merge pictures are presented (C,F,I). Note that the integral Golgi protein d120kd is redistributed back to the ER upon treatment of S2 cells with BFA (nuclear envelope staining, arrows), similar to the behaviour of the Golgi enzymes in BFA-treated mammalian cells (Lippincott-Schwartz et al., 1989). Additionally, the focused organisation of the tER sites in BFA-treated S2 cells was also disrupted, whereas this was not the case in mammalian cells (studies following Sec13-YFP; Ward et al., 2001). Note that H89 treatment results in an extensive dispersal of dSec23p in the majority of the S2 cells, in agreement with the reported cytosolic redistribution of COPII coat subunits in H89-treated mammalian cells (Lee and Linstedt, 2000). Furthermore, the d120kd-positive spots appeared smaller and dispersed. Incubation of control cells at 37°C in C does not affect the organisation of the early exocytic pathway (compare with Figure 7C). Bars: 5μm.
• Supplemental Figure 4 - Golgi stacks and clusters in dGRASP/dGM130 depleted cells are equally active in Delta anterograde transport.
  S2 cell depleted of dGRASP and dGM130 for 96h was labelled for Delta (10nm) and p24δ1 (15nm). Both the remaining Golgi stack (B) and the Golgi cluster (C) are labelled to the same extent with anterograde marker Delta. N, nucleus; PM, plasma membrane; E, endosomes. Bars: 200nm.
• Supplemental Figure 5 - dGRASP depletion does not affect retention of p24δ1 in the early exocytic compartments
  (A-B) S2 cells were processed for IEM, and labelled for p24δ1 (10nm gold) (A) or double labelled for p24δ1 (15nm gold) and dSec23p (10nm gold) (B). The majority of p24δ1 labelling is confined to the pleiomorphic tER membranes.(C-D) Mock-treated (C) and dGM130/dGRASP depleted (D) S2 cells for 96h were labelled for p24δ1 (green) and d120kd (red). Note that the partial overlap of the p24δ1 with the Golgi marker does not increase upon dGRASP/dGM130 depletion and no trace amount of protein is detected at the plasma membrane, as it would be expected if dGRASP was involved in its retention to tER/cis-Golgi interface. G, Golgi stacks; ER, endoplasmic reticulum; tER, transitional ER; NE, nuclear envelope. Bars: 200nm (A-B) and 5μm (C-D).