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First published online 23 November 2004
doi: 10.1242/jcs.01542

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Multiple regions contribute to membrane targeting of Rab GTPases

¹ Cell and Molecular Biology, Division of Biomedical Sciences, Faculty of Medicine, Imperial College London, London, SW7 2AZ, UK
² Department of Basic Medical Sciences, St George's Hospital Medical School, London, SW17 0RE, UK
³ Texas A&M University, College Station, TX 77843, USA

View larger version (34K): [in a new window]   Fig. 1. Schematic representation of wild-type and C-terminal hybrid Rab proteins used in this study. The numbers above each bar are residue numbers indicating the total length of each wild-type Rab, or the point at which the hypervariable region from another Rab was fused to generate a hybrid protein. On the right, the subcellular localisations of the proteins used are summarised: SG, secretory granule; EE, early endosome; G, Golgi; *denotes partial mislocalisation.

View larger version (61K): [in a new window]   Fig. 2. Expression of EGFP-Rab27/Rab5 fusion proteins in AtT20 cells. Cells were transiently transfected with EGFP-Rab27 (A-C), EGFP-Rab5Q79L (D-F), EGFP-Rab5Q79L182/Rab2739 (G-L) or EGFP-Rab27187/Rab535 (M-R). After fixation, cells were permeabilised, and immunolabelled with antibodies to ACTH (B,H,N) or Tfn-R (E,K,Q). Panels on the left (A,D,G,J,M,P) show EGFP fluorescence and on the right (C,F,I,L,O,R) are the merged images.

View larger version (63K): [in a new window]   Fig. 3. Expression of EGFP-Rab27/Rab1 fusion proteins in AtT20 cells. Cells were transiently transfected with EGFP-Rab1 (A-C), EGFP-Rab1172/Rab2740 (D-I) or EGFP-Rab27187/Rab134 (J-O). After fixation, cells were permeabilised and immunolabelled with antibodies to ACTH (E,K) or the Golgi 58K protein (B,H,N). Panels on the left (A,D,G,J,M) show EGFP fluorescence and panels on the right (C,F,I,L,O) are the merged images.

View larger version (76K): [in a new window]   Fig. 4. Expression of EGFP-Rab proteins in ashen melanocytes. Melan-ash2 cells were transiently transfected with EGFP-Rab27 (C,D), EGFP-Rab5 (E,F), EGFP-Rab1 (G,H), EGFP-Rab27187/Rab535 (I,J) or EGFP-Rab27187/Rab134 (K,L). A and B show non-transfected cells; A,C,E,G,I,K show EGFP fluorescence, and B,D,F,H,J,L show the corresponding phase-contrast images.

View larger version (101K): [in a new window]   Fig. 5. Transient over-expression of EGFP-hRab5a and lower eukaryote (S. cerevisiae and T. brucei) homologues in HeLa cells. HeLa cells were transfected with EGFP-hRab5a (A-C), EGFP-Ypt51 (D-F), EGFP-Ypt52 (G-I), EGFP-Ypt53 (J-L), EGFP-TbRab5A (M-O) or EGFP-TbRab5B (P-R) After 24 hours, cells were fixed, permeabilised and immunostained with an anti-Tfn-R antibody (B,E,H,K,N,Q) as described in Materials and Methods. The panels on the left (A,D,G,J,M,P) are EGFP fluorescence and those on the right (C,F,I,L,O,R) are the merged fluorescent signals.

View larger version (83K): [in a new window]   Fig. 6. Transient over-expression in HeLa cells of EGFP-Rab5 fusion proteins containing the hypervariable domain of other Rab proteins. HeLa cells were co-transfected with C-myc-Rab5a and EGFP-Rab5175/Rab139 (A-C), EGFP-Rab5174/Rab252 (G-I) or EGFP-Rab5174/Rab741 (M-O). Cells transfected with the constitutively active forms of the hybrid proteins EGFP-Rab5QL175/Rab139 (D-F), EGFP-Rab5QL174/Rab252 (J-L) and EGFP-Rab5QL174/Rab741 (P-R) were immunostained with anti-EEA1 antibody (E,K,Q).

View larger version (72K): [in a new window]   Fig. 7. Transient over-expression in HeLa cells of several EGFP-Rab fusion proteins containing the hypervariable domain of Rab5a. HeLa cells were transfected with EGFP-Rab1a (A-C), EGFP-Rab1156/Rab550 (D-F), EGFP-Rab2 (G-I) or EGFP-Rab2160/Rab541 (J-O). After 24 hours, cells were fixed, permeabilised and immunostained with an anti-Golgin97 antibodies (B,E,H,K), or Tfn-R antibody (N) as described in Materials and Methods. The panels on the left (A,D,G,J,M) show the EGFP signal and on the right (C,F,I,L,O) are the merged fluorescent signals.

View larger version (89K): [in a new window]   Fig. 8. Transient over-expression in HeLa cells of EGFP-Rab5Q79L with the RabF or RabSF domains of Rab27a. HeLa cells were transfected with EGFP-Rab5QL/Rab27SF3 (A-I), EGFP-Rab5QL/Rab27F4 (J-L), EGFP-Rab5QL/Rab27SF1-SF4 (M-O) or EGFP-Rab5QL/Rab27SF2 (P-R). After 24 hours, cells were fixed, permeabilised and immunostained with an anti-Tfn-R) antibody (B,K,N,Q), with anti-PDI antibodies (E) or anti-Golgin97 antibodies (H). The panels on the left (A,D,G,J,M,P) are EGFP signal and on the right (C,F,I,L,O,R) are the merged fluorescent signals.

View larger version (62K): [in a new window]   Fig. 9. Expression of chimeric EGFP-Rab27 containing RabSF or RabF domains of Rab5 in AtT20 cells. Cells were transiently transfected with EGFP-Rab27/Rab5SF2 (A-F), EGFP-Rab27/Rab5SF3 (G-L) or EGFP-Rab27/Rab5F4 (M-O). After fixation, cells were permeabilised and immunolabelled with antibodies to ACTH (B,H,N) or the Golgi 58K protein (E,K). Panels on the left (A,D,G,J,M) show EGFP fluorescence and panels on the right (C,F,I,L,O) are the merged images.

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