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First published online 19 October 2004
doi: 10.1242/jcs.01491

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Localization of GDP-mannose transporter in the Golgi requires retrieval to the endoplasmic reticulum depending on its cytoplasmic tail and coatomer

Department of Biotechnology, University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan

View larger version (50K): [in a new window]   Fig. 1. GMT recycles between the ER and Golgi apparatus. (A,B) Yeast cells producing the Myc6-tagged GMT, Anp1p or Van1p from CEN plasmid were fixed with formaldehyde at the time indicated after the shift to 37°C and prepared for indirect immunofluorescence staining using anti-Myc monoclonal antibody 9E10 and FITC-conjugated affinity-purified goat anti-mouse-IgG. Cells were treated with cycloheximide (0.1 mg ml–1) before being shifted to 37°C. After 20 minutes at 37°C, the culture was divided in two; one portion was kept at 37°C and the other was shifted back to 25°C and incubated for 20 minutes to see the reversibility of localization (Recovery). (A) Wild-type (WT) and sec18ts mutant cells expressing Myc6-GMT. (B) Wild-type and sec23ts mutant cells expressing Myc6-GMT, Myc6-Anp1p and Myc6-Van1p. (C) Subcellular fractionation of the wild type and sec23-mutant-expressing Myc6-GMT. Medium-speed pellet (P7), high-speed pellet (P100) and high-speed supernatant (S100) were prepared, and GMT was detected by immunoblotting.

View larger version (74K): [in a new window]   Fig. 2. GMT is precipitated with coatomer. The cleared lysate was prepared from yeast producing the Myc6-tagged protein of interest. The tagged protein was immunoprecipitated with anti-Myc antibody and Protein-A/Sepharose beads. Each sample was separated by SDS-PAGE and analysed by immunoblotting with anti-Myc or anti-Ret2p antibody. The total, unbound and bound fractions were examined for Myc6-GMT (pMAV426; lanes 1-3), Myc6-GMT4 (pMAV424; lanes 4-6), Myc6-GMT12 (pMAV425; lanes 7-9), GMT-Myc6 (pMA12; lanes 10-12), GMT12-Myc6 (pMA50; lanes 13-15) and Anp1p-Myc6 (pSV314; lanes 16-18).

View larger version (139K): [in a new window]   Fig. 4. Subcellular localization of the GFP-GMT tail mutants in the S. cerevisiae cells by indirect immunofluorescence. The GFP-tagged GMT tail mutant genes were expressed from the original VIG4 promoter on a CEN ARS plasmid in the wild-type cell. The cells were fixed with formaldehyde and observed by indirect immunofluorescence using anti-GFP antibody. The C-terminal sequences are shown. For expression of GFP-tagged GMT mutant derivatives, the plasmids used were pMA89 (A), pMA86 (B), pMAV587 (C), pMAV594 (D), pMAV595 (E), pMAV597 (F), pMAV599 (G) and pMAV601 (H).

View larger version (40K): [in a new window]   Fig. 3. The coatomer subunit Ret2p binds to the GST-fusion peptides related to the cytoplasmic tail of GMT. The GST-fusion proteins were produced in E. coli and affinity purified by glutathione-Sepharose beads. The yeast lysate was prepared from the cells producing the 3HA-tagged Ret2p with glass beads and 0.5% Triton X-100 and used as the labelled coatomer. The beads were mixed with the lysate, incubated for 16 hours at 4°C and washed extensively, and the bound proteins were analysed by SDS-PAGE. The bound Ret2p was detected by immunoblot analysis with 12CA5 (top). The amount of GST-fusion peptide was shown by Coomassie Brilliant Blue staining (bottom). In lanes 1 and 14, the purified GST was used as the negative control. In lanes 2 and 15, the GST-fusion protein containing the Wbp1p peptide was used as the positive control. In lane 3, the GST-fusion protein containing the wild-type GMT C-terminal peptide was used.

View larger version (48K): [in a new window]   Fig. 6. Subcellular localization of the GFP-GMT-Wbp1p chimeric protein, which exchanges the C-terminal peptide of the GMT tail for that of Wbp1p. This chimeric protein is tagged with GFP and produced by VIG4 promoter and CEN ARS plasmid (pMAV592) in the wild-type cell. (A) The cells were fixed with formaldehyde and observed by indirect immunofluorescence using anti-GFP antibody (left) or by phase contrast microscopy (right). (B) Activity staining of invertase in 7.5% SDS-PAGE of the periplasmic fraction of the wild-type and vig4-1 transformant cells.

View larger version (82K): [in a new window]   Fig. 5. The sec21ts mutant cells expressing a GFP-tagged GMT. The sec21ts mutant cells expressing a GFP-tagged GMT were treated with cycloheximide (0.1 mg ml–1) before being shifted to 37°C. The cells were fixed with formaldehyde and observed by indirect immunofluorescence using anti-GFP antibody (IF) and by phase-contrast microscopy (PC).

View larger version (77K): [in a new window]   Fig. 7. Localization of the mutant Emp47p with modified C-terminus. Cells were stained by immunofluorescence using anti-Myc and anti-Mnn9 antibodies to visualize Emp47p (myc-Emp47) and the Golgi mannosyltransferase (Mnn9), respectively. Merged images for phase-contrast microscopy are shown at the right. (A) The wild-type Emp47p (-RIRQEIIKTKLL). (B) A truncated Emp47p (-RIRQEII). (C) Addition of the GMT C-terminal peptide to the truncation (-RIRQEIIGSKQKKQQAQPLRK). (D) A C-terminal replacement with the GMT C-terminal peptide (-GSKQKKQQAQPLRK).