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First published online December 15, 2003
doi: 10.1242/10.1242/jcs.00858

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Activity of recycling Golgi mannosyltransferases in the yeast endoplasmic reticulum

¹ Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland
² Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland

View larger version (32K): [in a new window]   Fig. 1. 1,6-Mannosylation of pro-CPY in the ER. Sec13-1 (lanes 1 and 2; H230), sec18-1 (lanes 3; H4), sec23-1 (lanes 4; H238) and och1 (panel C, H1691) were preincubated for 15 minutes, pulse-labelled with [35S]methionine/cysteine for 5 minutes and chased in the presence of CHX for 30 minutes (A,B) or 20 minutes (C) at the indicated temperatures. The cell lysates were subjected to immunoprecipitation with CPY antiserum alone, or first with CPY antiserum followed by reimmunoprecipitation with antiserum against 1,6-mannose residue as indicated, followed by SDS-PAGE analysis. The untranslocated form (pre), ER form (p1), Golgi form (p2) and mature CPY (m) are indicated on the right.

View larger version (57K): [in a new window]   Fig. 2. 1,6-Mannosylation of bulk glycoproteins in the ER. Sec13-1 (H230), sec23-1 (H481) and och1 (H1691) cells were preincubated for 15 minutes and 35S-labeled for 30 minutes at 37°C. The lysed cells were precipitated with concanavalin A-Sepharose. Half of the precipitates were subjected to SDS-PAGE directly (uneven lanes). The proteins of the other half of the samples were released from the lectin beads and immunoprecipitated with 1,6-mannose antiserum (even lanes).

View larger version (34K): [in a new window]   Fig. 3. Elongation of O-glycans of Hsp150-ß-lactamase in the ER. Sec13-1 (lanes 1-12; H1065), or sec18-1 (lanes 13-16; H393) mutants were preincubated for 15 minutes, pulse labelled for 5 minutes with [35S]methionine/cysteine (lanes 1-8 and 13-16) or [3H]mannose (lanes 9-12), and chased with CHX as indicated. The cell lysates (c; lanes 1-4 and 9-16) and culture media (m; lanes 5-8) were subjected to immunoprecipitation with ß-lactamase antiserum followed by SDS-PAGE analysis. The migration of the various Hsp150-ß-lactamase forms is indicated on the right, and that of molecular weight markers on the left.

View larger version (32K): [in a new window]   Fig. 4. Electrophoretic migration of Hsp150-ß-lactamase-HDEL. Normal cells expressing Hsp150-ß-lactamase-HDEL (H606; lanes 1 and 2) or Hsp150-ß-lactamase (H335; lanes 3 and 4), and a sec18-1 mutant expressing Hsp150-ß-lactamase-HDEL (H610; lanes 5 and 6) were preincubated for 15 minutes and 35S-labeled for 30 minutes at 37°C. The medium (m) and respective cell lysate (c) samples were immunoprecipitated with ß-lactamase antiserum and analysed by SDS-PAGE. The figures on the right indicate biosynthetic intermediates of the reporter proteins and those on the left molecular weight markers.

View larger version (27K): [in a new window]   Fig. 5. Scw4p glycosylation in a COPI mutant. Control (H1495), sec23-1 (H1496) and sec21-1 (H1497) cells were preincubated at 37°C for 15 minutes, and pulse-labelled with [35S]methionine/cysteine for 5 minutes. Parallel samples were chased with CHX for 30 minutes, as indicated. TM was present from the preincubation onwards, as indicated. The cell lysates were immunoprecipitated with antibody against pentahistidine only (lanes 1-12), or reimmunoprecipitated with antiserum against 1,6-mannose (lanes 13-15), followed by SDS-PAGE analysis. Apparent molecular weights of Scw4p forms are indicated on the right.

View larger version (98K): [in a new window]   Fig. 6. Immunofluorescent staining of Och1p-HA in the sec23-1 mutant. (A,B,E,F) Och1p-HA in sec23-1 (H1490) or (C,D) Och1p-HA plus cytochrome b(5)-opsin in sec23-1 (H1791) were grown at 24°C, followed by a 1 hour incubation with CHX at 37°C. The cells were fixed and immunostained with polyclonal antibody against HA (A,C,E). The cell samples on the right were viewed through Nomarski optics (B), or double stained with monoclonal antibody against opsin (D), or with DAPI (F). The arrows point to ER-like structures (C,D,E) or nuclei (F).

View larger version (80K): [in a new window]   Fig. 7. Immunofluorescent staining of Och1p-HA. Sec23-1 cells (H1490; A,B), sec7-1 cells (H1489; C), or normal cells (H1488; D) were grown at 24°C and fixed immediately (A,D), or incubated with CHX for 1 hour at 24°C (B), or thereafter for an additional 30 minutes at 37°C (C) before fixation. The cells were subjected to indirect immunofluorescent staining with antibody against the HA epitope (left), and DAPI (right).

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