Subcellular fractionation of actively growing protoplasts of Saccharomyces cerevisiae |
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Authors: | JoséP. Martínez Amelia Murgui Alberto Flores Rafael Sentandreu |
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Affiliation: | Departamento de Microbiología, Facultad de Farmacia, Universidad de Valencia, Avenida Blasco Ibañez 13, Valencia 10 Spain |
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Abstract: | Cell homogenates obtained from partially regenerated Saccharomyces cerevisiae protoplasts were fractionated by a procedure using a combination of continuous and discontinuous sucrose gradients, under experimental conditions that minimize possible artifacts due to centrifugation and resuspension. At least five different membranous organelle fractions (plasma membrane, mitochondria, rough endoplasmic reticulum, smooth endoplasmic reticulum-like structures and small-sized particulated structures) were isolated. Subcellular fractions were characterized by assaying established marker enzymes. Radioactive labelled ([U-3H]uracil) ribosomes were also used as a further characterization criterion of the rough endoplasmic reticulum. Comparative SDS-polyacrylamide gel electrophoresis of the protein constituents of the isolated membrane-bound organelles suggest that the polypeptide pattern could also be used as an additional marker for some of these structures. Finally, subcellular distribution of chitin synthase was determined using this fractionation procedure, and two partially zymogenic enzyme pools (one inside the cell associated to particles which sediments at high speed, and the second one associated to the plasma membrane) were found. |
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Keywords: | Subcellular fractionation Protoplast Chitin synthase Polypeptide profile (S. cerevisiae) |
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