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First published online 15 July 2003
doi: 10.1242/jcs.00641

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Evidence that the transport of ricin to the cytoplasm is independent of both Rab6A and COPI

The Molecular and Cell Biology Department, FO31, The University of Texas at Dallas, Box 830688, Richardson, TX 75083-0688, USA

View larger version (16K): [in a new window]   Fig. 3. The effect of Rab6A-T27N on the rate at which ricin first enters the cytoplasm. Mock-transfected control cells () or cells transfected with Rab6A-T27N () for 18 hours were chilled and incubated with 100 µg/ml of ricin for an hour to bind the toxin to cell surface receptors. The cells were raised to 37°C by addition of warm medium and protein synthesis was assessed at the indicated times as described in Materials and Methods. The solid (control) and broken (transfected with Rab6A-T27N) lines were fitted to the data points by the method of least squares.

View larger version (78K): [in a new window]   Fig. 1. The effect of Rab6A-T27N on the transport of HA to the plasma membrane in Vero cells. Vero cells were transfected with or without Rab6A-T27N for 18 hours, infected with influenza virus, radiolabeled and then chased for 60 minutes as described in Materials and Methods. After trypsin treatment, the cells were lysed and HA proteins were immunoprecipitated and resolved in a 12% SDS-polyacrylamide gel. Transport to the plasma membrane was determined by trypsin sensitivity as described in Materials and Methods. HA0 is intact HA whereas HA1 and HA2 are the large and small trypsin fragments, respectively.

View larger version (17K): [in a new window]   Fig. 2. The effect of Rab6A-T27N on the sensitivity of cells to Shiga toxin and ricin. Vero cells were either mock transfected () or transfected with a plasmid encoding mutant Rab6A-T27N () for 18 hours and treated with various concentrations of Shiga toxin (A) or ricin (B) for 2.5 hours. Tran 35S-label was added for 30 minutes and the incorporation of radioactivity into acid-insoluble material was determined as described in Materials and Methods. Each point is the average of three independent experiments.

View larger version (84K): [in a new window]   Fig. 4. The distribution of TGN-38 and Rab6A-GFP in ldlF cells. Cells were transfected with plasmid DNA encoding Rab6A-GFP and incubated for 48 hours at 34°C. Cells were left at 34°C (A and B) or shifted to 39.5°C for 6 hours (C and D). The cells were then fixed and stained with anti-TGN-38 followed by a rhodamine-labeled secondary antibody. (A) and (C) show rhodamine-labeled anti-TGN-38 and B and D are direct visualization of GFP in the fields shown in A and C, respectively. The arrows in C and D show examples of structures that label with both TGN-38 and Rab6A-GFP in ldlF cells at the high temperature. Scale bar in D: 25 µm.

View larger version (27K): [in a new window]   Fig. 5. Rab proteins involved in retrograde transport from endosomes to the ER. Toxins enter cells either by clathrin-independent or clathrin-dependent endocytosis. Initial pathways of uptake converge on early endosomes, except for the caveosome pathway which apparently bypasses the typical endosomal system in transporting material to the ER. One pathway from early endosomes to the TGN is via late endosomes that use Rab7 and Rab9 in sequential steps. Another pathway to the TGN is via recycling endosomes and involves Rab6A' and Rab11 in different steps. Rab6A' may also participate in the direct transport of material from early endosomes to the TGN. Two pathways are proposed to transport material from the TGN-Golgi complex to the ER, the COPI-dependent pathway that is regulated by Rab6A and the COPI-independent pathway that is not believed to require Rab6A.