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First published online 25 August 2004
doi: 10.1242/jcs.01349

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Gyp5p and Gyl1p are involved in the control of polarized exocytosis in budding yeast

Laurent Chesneau^1,*, Sophie Dupré^1,*, Anna Burdina¹, Jérôme Roger^1,, Sophie Le Panse², Michel Jacquet¹ and Marie-Hélène Cuif^1,¶

¹ Institut de Génétique et Microbiologie, CNRS-UMR 8621, Université Paris XI, 91 405 Orsay Cédex, France
² Service de Microscopie Electronique, Institut J. Monod, 75251 Paris CEDEX 05, France

View larger version (57K): [in a new window]   Fig. 1. Gyp5p and Gyl1p localize at the bud tip during polarized growth, at the bud neck during cytokinesis, and are partially colocalized. (A) Yeast cells expressing Gyp5p-Myc (green) and Gyl1p-HA (red) were stained by indirect immunofluorescence and observed with a 3D deconvolution microscopy system. Images shown are projections of deconvoluted Z-series combining green and red signals. Bars, 1 µm. White squares indicate the regions magnified in B. (B) Single sections of part of each image in A. Bars, 1 µm.

View larger version (42K): [in a new window]   Fig. 2. (A) Gyp5p and Gyl1p do not colocalize with actin. Yeast cells expressing Gyl1p-HA or Gyp5p-HA were stained by immunofluorescence and examined with a 3D deconvolution microscopy system. Images are projections of deconvoluted Z-series combining green, red and blue signals. Bars, 1 µm. First row: Gyl1p-HA is stained in red, actin is stained in green, DNA is stained in blue. Second row: Gyp5p-HA is stained in red, actin is stained in green, DNA is stained in blue. (B) Gyp5p-Myc and Gyl1p-HA localize within the septin ring. Yeast cells expressing Gyl1p-HA and Gyp5p-Myc were stained by immunofluorescence and examined with a 3D deconvolution microscopy system. Images are projections of deconvoluted Z-series combining green, red and blue signals. Cdc11p is stained in green, Gyl11p-HA is stained in red and Gyp5p-Myc is stained in blue. The magenta signal is the result of combination of red and blue signals. Bud emergence and post-cytokinesis stages are shown. Bars, 1 µm.

View larger version (45K): [in a new window]   Fig. 3. (A) Gyp5p and Gyl1p are present in three main cellular pools: yeast cells expressing Gyp5p-Myc, Gyl1p-HA and Sec4p-GFP were submitted to subcellular fractionation to generate P13, P100 and S100 fractions. The volumes of fractions loaded on each lane of SDS-PAGE correspond to 2x107cells. The nitrocellulose membranes were incubated with either anti-Myc, anti-HA or anti-GFP antibodies, then stripped from the antibodies and incubated again with anti-Pma1p antibodies (from R. Serrano) and anti-actin antibodies. (B) Gyp5p and Gyl1p co-fractionate with plasma membrane and vesicles markers: the P13 and P100 fractions shown in A were loaded on linear 20-60% sucrose gradients. Fractions were probed by immunoblotting with different antibodies as described in A. Dpm1p is an ER membrane dolichol-phosphate mannose synthase. (C) Gyp5p and Gyl1p behave as peripheral membrane proteins: yeast cells expressing Gyp5p-Myc and Gyl1p-HA were submitted to membrane extraction, as described in Materials and Methods. S, supernatant; P, membrane pellet. (D) Gyp5p and Gyl1p are phosphorylated proteins: total protein extracts of yeast cells expressing Gyp5p-Myc and Gyl1p-HA were submitted to calf intestine alkaline phosphatase (CIP) treatment, as described in Material and Methods, resolved on SDS-PAGE and revealed with either anti-Myc or anti-HA antibodies.

View larger version (22K): [in a new window]   Fig. 4. Gyl1p-HA co-immunoprecipitates with Gyp5p-Myc. Volumes of subcellular fractions corresponding to 5x107 yeast cells expressing Gyp5p-Myc and Gyl1p-HA were submitted to immunoprecipitation with anti-Myc antibodies. Proteins bound to Protein G-agarose beads (Pellet) or remaining in the supernatant (Supernatant) were separated on SDS-PAGE, and revealed with either anti-Myc or anti-HA antibodies. One fifth of the total amount of proteins was loaded in supernatant lanes.

View larger version (57K): [in a new window]   Fig. 5. Gyp5p-Myc and Gyl1p-HA interact with Sec4p-GFP. Lane 1, bud emergence; lane 2, small-budded cell; lane 3, cytokinesis. (A) Subcellular fractions obtained from 108 cells expressing Gyp5p-Myc, Gyl1p-HA and Sec4p-GFP were submitted to immunoprecipitation with anti-GFP antibodies, as described in Materials and Methods. Proteins bound to the agarose beads (Pellet), or remaining in the supernatant (Supernatant), were separated on SDS-PAGE and revealed with either anti-Myc, anti-HA or anti-GFP antibodies. One fifth of the total amount of proteins was loaded in supernatant lanes. In the control lanes, anti-GFP antibodies were omitted. (B) Yeast cells expressing Gyp5p-Myc, Gyl1p-HA and Sec4p-GFP were stained by immunofluorescence and examined with 3D deconvolution microscopy. Gyl1p-HA is stained in red, Sec4p-GFP is stained in green and Gyp5p-Myc is stained in blue. Images in the first row are projections of deconvoluted Z-series combining red, green and blue signals. White squares indicate the region magnified in the second row. Images in the second row are single sections shown with higher magnification. Bars, 1 µm.

View larger version (28K): [in a new window]   Fig. 6. Overexpression of Gyp5p-Myc enhances the sec2-78 phenotype. An YCpADH1 vector, empty or carrying Myc-tagged Gyp5p, was transformed into a sec2-78 strain. Successive dilutions of transformed cells in exponential growth phase were spotted onto selective medium and incubated for 2 days at 26°C, 30°C or 37°C.

View larger version (48K): [in a new window]   Fig. 7. gyp5gyl1 cells display a cold-sensitive slow growth phenotype and a frequent accumulation of secretory vesicles in small buds. (A) Doubling time of WT and gyp5gyl1 strains in rich medium was determined in early log phase at 13° and 30°C. Values are the average of results obtained with four independent clones. (B) Thin section electron microscopy was performed on WT and gyp5gyl1 cells cultured at 13°C for 16 hours. Small buds correspond to a ratio (bud width/bud neck width) <2, large buds to a ratio of 2 and higher. The % of WT cells and gyp5gyl1 cells containing at least five distinct secretory vesicles were determined. (C) Thin section electron microscopy of WT (a) and gyp5gyl1 cells (b-d). Bars, 500 nm.

View larger version (36K): [in a new window]   Fig. 8. gyp5gyl1 cells display a distal secretion defect at 13°C. (A) Invertase expression was induced in WT and gyp5gyl1 cells cultured at 13°C, and invertase secretion was monitored as described in Materials and Methods. The % of secreted invertase is the ratio (external invertase/total invertase). Values are the average of three experiments performed on two independent clones. One invertase unit is defined as the enzyme quantity producing 1 µmol of glucose/minute/107 cells. (B) Proximal steps of secretory pathway are normal in gyp5gyl1 cells. Pulse-chase labelling and immunoprecipitation of CPY was performed on WT and gyp5gyl1 cells cultured at 13°C, as described in Materials and Methods. The ER (p1), Golgi (p2) and mature (m) forms of CPY are indicated. (C) Bgl2p-HA secretion assay was monitored as described in Materials and Methods. Equal amounts of cells were loaded on SDS-PAGE. After separation, proteins in the top of the gel were stained by Coomassie blue and proteins of the bottom of the gel were transferred onto nitrocellulose membrane. Immunodetection of this membrane with HA antibody was then performed. The control is one protein of the top of the gel stained by Coomassie blue.