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The yeast ADP-ribosylation factor GAP, Gcs1p, is involved in maintenance of mitochondrial morphology

Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan

View larger version (37K): [in a new window]   Fig. 1. Maturation of vacuolar enzymes ALP and CPY. Wild-type (ARF1/GCS1) and gcs1 and arf1 mutant cells were grown, radiolabeled with 35S-labeled methionine and cysteine and incubated for the indicated chase time (min) before immunoprecipitation. p1 CPY is the core-glycosylated form found in the ER, p2 is the outer chain-glycosylated Golgi form and the mature form (M) results from proteolytic processing in the vacuole. pALP is the proenzyme form and mALP is the mature form in the vacuole.

View larger version (63K): [in a new window]   Fig. 2. Detection of Gcs1p and mutated forms in yeast by western blot analysis. The indicated amounts of proteins from (A) YPH 252 wild-type or gcs1 mutant cells and (B) purified His-tagged Gcs1p or (C) 20 µg of proteins from gcs1 mutant cells overexpressing wild-type Gcs1p, HA-tagged zinc-finger-mutated (Zn) Gcs1p, PH domain-deleted (PH) Gcs1p or GFP-fused PH domain (GFP-PH) were separated by SDS-PAGE. Proteins were stained with Coomassie blue (A) or transferred to PVDF, reacted with affinity-purified antibodies against Gcs1p and detected using the ECL system. Positions of protein standards (kDa) are at the left.

View larger version (64K): [in a new window]   Fig. 3. Subcellular localization of endogenous Gcs1p. Yeast YPH 252 cells were grown in minimal medium (A) or YPD medium (B). Lysates of spheroplasts from cells were fractionated by sucrose gradient (10-60%) centrifugation. Proteins in samples (100 µl) of fractions were precipitated, separated by SDS-PAGE and analyzed by immunoblotting. Gcs1p, yARF1, Emp47 (Golgi marker), porin (mitochondrial marker), Kar2 (an ER marker), and PGK (cytoplasmic marker) were identified with specific antibodies and detected using the ECL system with exposure to Hyper-film MP. Gradient fractions were numbered from the top. Below the immunoblots in each panel are results of indirect immunofluorescence staining of YPH 252 wild-type (a, b, c, d) or gcs1 mutant (e) cells. Cells were fixed with formaldehyde; spheroplasts were prepared and reacted with purified anti-Gcs1p antibodies (c) or anti-porin antibody (d, e) followed by secondary antibodies. In (a) are phase images of the cells in b, c, and d. Nucleic acids were stained with H33258 (b). Arrows indicate perinuclear localization and arrowheads mitochondria.

View larger version (55K): [in a new window]   Fig. 4. Effect of overexpression of Gcs1p on mitochondrial morphology. Data are presented as in Fig. 3. (A) lysates of spheroplasts from gcs1 cells overexpressing wild-type recombinant Gcs1p under the control of the ADH1 promoter and grown in minimal medium were fractionated for analysis by immunoblotting. (B) and (C) indirect immunofluorescence staining of gcs1 cells overexpressing wild-type Gcs1p. (B) spheroplasts were reacted with purified anti-Gcs1p antibodies (b) or anti-porin antibody (c) or stained with H33258 (a). Arrows indicate perinuclear localization and arrowheads mitochondria. Panel C, spheroplasts were reacted with purified anti-Gcs1p antibodies (b) or anti-actin antibody (c) or stained with H33258 (a). Arrowheads indicate colocalization of Gcs1p and actin structures.

View larger version (22K): [in a new window]   Fig. 5. Gcs1p is weakly associated with membrane fraction. The ER/mitochondria-enriched fraction (P13) from wild-type yeast was treated with 1 M NaCl, 0.1 M Na2CO3 or buffer (Mock) on ice for 30 minutes, followed by centrifugation at 150,000 g for one hour. The resulting supernatant and pellet fractions were analyzed by western blotting using anti-Gcs1p or anti-porin antibodies.

View larger version (52K): [in a new window]   Fig. 6. Effect of overexpression of Gcs1p on mitochondrial morphology does not require its zinc-finger motif. Data are presented as in Fig. 3. Upper panel, lysates of spheroplasts from gcs1 cells overexpressing Gcs1Zn and grown in minimal medium were fractionated for analysis by immunoblotting. Lower panel, indirect immunofluorescence staining of gcs1 cells overexpressing Gcs1Zn. Spheroplasts were reacted with purified anti-Gcs1p antibody (B) or anti-porin antibody (C) or stained with H33258 (A). Arrows indicate tubular mitochondria and arrowheads mitochondria.

View larger version (59K): [in a new window]   Fig. 7. The C-terminal PH domain of Gcs1p is required for mitochondrial localization. Data are presented as in Fig. 3. Upper panel, lysates of spheroplasts from gcs1 cells overexpressing Gcs1PH and grown in minimal medium were fractionated. Lower panel, indirect immunofluorescence staining of spheroplasts reacted with purified anti-Gcs1p antibody (B) or anti-porin antibody (C) or stained with H33258 (A).

View larger version (79K): [in a new window]   Fig. 8. Overexpression of GFP-fused PH domain of Gcs1p did not affect distribution of mitochondrial or ER markers or actin. Immunofluorescence staining of gcs1 cells overexpressing GFP-fused PH domain and reacted with purified anti-Gcs1p antibody (c, g and k), anti-porin antibody (d), anti-Kar2 antibody (h) or Alexa FluorTM 594-phalloidin (l), or stained with H33258 (b, f and j).