Summary
Methods are described for isolating and identifying subcellular membranes from walled hyphae ofGilbertella persicaria. Differences in thickness and symmetry of membranes and in contents of vesicles were used to distinguish different types of membranes. Mitochondria, vacuoles, plasma membrane, and vesicles with attached ribosomes from homogenized germlings equilibrated at the 1.2/1.4 M interface in discontinuous sucrose gradients. Accelerated flotation in centrifuged Ficol-sucrose gradients resulted in the additional separation of the mixed membranes into three fractions: one contained predominantly intact mitochondria, another was composed of vacuoles and vesicles coated with ribosomes, and a third was enriched in plasma membranes. Based upon morphometric analysis, these fractions contained 92% mitochondria, 53% vacuoles, and 89% plasma membranes, respectively. The source of vesicles coated with ribosomes was investigated since rapidly growing hyphae ofG. persicaria contained little rough endoplasmic reticulum as compared with other classes of membranes. Reconstruction from electron micrographs of mitochondrial fragmentation and vesiculation suggested that most of the ribosome-coated vesicles originated from disrupted mitochondria rather than from rough endoplasmic reticulum. The study demonstrates the utility of ultrastructural markers to identify membranesin vitro independent of, or as an adjunct to, cytochemical and biochemical markers.
Similar content being viewed by others
References
Bachmann, B. J., Bonner, D. M., 1959: Protoplasts fromNeurospora crassa. J. Bacteriol.78, 550–556.
Bartnicki-Garcia, S., Nelson, N., Cota-Robles, E., 1968: Electron microscopy of spore germination and cell wall formation inMucor rouxii. Arch. Mikrobiol.63, 242–255.
Bracker, C. E., 1968: The ultrastructure and development of sporangia inGilbertella persicaria. Mycologia60, 1016–1067.
—, 1971: Cytoplasmic vesicles in germinating spores ofGilbertella persicaria. Protoplasma72, 381–397.
—,Ruiz-Herrera, J., Bartnicki-Garcia, S., 1976: Structure and transformation of chitin synthetase particles (chitosomes) during microfibril synthesisin vitro. Proc. Natl. Acad. Sci. USA73, 4570–4574.
—,Grove, S. N., 1971: Continuity between cytoplasmic endomembranes and outer mitochondrial membranes in fungi. Protoplasma73, 15–34.
Cabib, E., Ulane, R., Bowers, B., 1973: Yeast chitin synthetase. J. biol. Chem.248, 1451–1458.
Duran, A., Bowers, B., Cabib, E., 1977: Chitin synthetase zymogen is attached to the yeast plasma membrane. Proc. Natl. Acad. Sci. USA72, 3952–3955.
Douce, R., Manella, C. A., Bonner, W. D., 1973: The external NADH dehydrogenases of intact plant mitochondria. Biochim. biophys. Acta292, 105–116.
Fuhrmann, G. F., Wehrli, E., Boehm, C., 1974: Preparation and identification of yeast plasma membrane vesicles. Biochim. biophys. Acta363, 295–310.
Garcia-Acha, I., Lopez-Belmonte, F., Villanueva, J. R., 1967: Vacuoles of fungal protoplasts. Canad. J. Microbiol.13, 433–437.
Grove, S. N., Bracker, C. E., Morré, D. J., 1968: Cytomembrane differentiation in the endoplasmic reticulum-Golgi apparatus-vesicle complex. Science (N.Y.)161, 171–173.
Hardy, B., Bensch, K. G., Schrier, S. F., 1979: Spectrin rearrangement early in erythrocyte ghost endocytosis. J. Cell Biol.82, 654–663.
Hoch, H. C., Howard, R. J., 1980: Ultrastructure of freeze-substituted hyphae of the BasidiomyceteLaetisaria arvalis. Protoplasma103, 281–297.
Hodges, T. K., Leonard, R. T., Bracker, C. E., Keenan, T. W., 1972: Purification of an ion-stimulated adenosine triphosphatase from plant roots: Association with plasma membranes. Proc. Natl. Acad. Sci. USA69, 3307–3311.
Indge, K. J., 1968: The isolation and properties of the yeast cell vacuoles. J. gen. Microbiol.51, 441–446.
Iten, W., Matile, P., 1970: Role of chitinase and other lysosomal enzymes ofCoprinus lagopus in the autolysis of fruiting bodies. J. gen. Microbiol.61, 301–309.
Kellems, R. E., Allison, V. F., Butow, R. A., 1975: Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria. J. Cell Biol.65, 1–14.
Leigh, R. A., Branton, D., 1976: Isolation of vacuoles from root storage tissue ofBeta vulgaris L. Plant Physiol.58, 656–662.
— —,Marty, F., 1979: Methods for the isolation of intact vacuoles and fragments of tonoplast. In: Plant Organelles (Reid, E., ed.) New York: J. Wiley and Sons.
Loud, A. V., 1962: A method for quantitative estimation of cytoplasmic structures. J. Cell Biol.15, 481–487.
Luft, J. H., 1961: Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol.9, 409.
Mahlberg, P. G., Turner, F. R., Walkinshaw, C., Veneketeswaren, S., 1974: Ultrastructural studies on plasma membrane related secondary vacuoles in cultured cells. Amer. J. Bot.61, 730–738.
Matile, P., 1966: Enzyme der Vakuolen aus Wurzelzellen von Maiskeimlingen. Ein Beitrag zur funktionellen Bedeutung der Vakuole bei der intrazellularen Verdauung. Z. Naturforsch.21 b, 871–878.
—, 1971: Vacuoles, lysosomes ofNeurospora. Cytobiologie3, 324–330.
—, 1978: Biochemistry and function of vacuoles. Ann. Rev. Plant Physiol.29, 193–213.
—,Wiemken, A., 1967: The vacuole as the lysosome of the yeast cell. Arch. Mikrobiol.56, 148–151.
— —, 1974: Vacuoles and spherosomes. Methods in Enzymol.31, 572–578.
Moore, T. S., Lord, J. M., Kagawa, T., Beevers, H., 1973: Enzymes of phospholipid metabolism in the endoplasmic reticulum of castor bean endosperm. Plant Physiol.52, 50–53.
Moreau, F., Lance, C., 1972: Isolement et propriétés des membranes externes et internes de mitochondries végétales. Biochimie54, 1335–1348.
Morré, D. J., 1971: Isolation of Golgi apparatus. Methods in Enzymol.22, 130–148.
—,Bracker, C. E., 1976: Ultrastructural alteration of plant plasma membranes induced by auxin and calcium ions. Plant Physiol.58, 544–547.
Nakamura, K. D., 1973: The isolation of vacuoles fromCandida utilis. Prep. Biochem.3, 553–561.
Osumi, M., Imaizumi, F., Imai, M., Sato, H., Yamaguchi, H., 1975: Isolation and characterization of microbodies fromCandida tropicalis PK 233 cells grown on normal alkanes. J. gen. appl. Microbiol.21, 375–387.
Peberdy, J. F., 1976: Isolation and properties of protoplasts from filamentous fungi. In: Microbial and Plant Protoplasts. (Peberdy, J. F., Rose, A. H., Rogers, H. J., Cocking, E. C., eds.), pp. 39–50. London: Academic Press.
Philipp, E. I., Franke, W. W., Keenan, T. W., Stadler, J., Jarasch, E. D., 1976: Characterization of nuclear membranes and endoplasmic reticulum isolated from plant tissue. J. Cell Biol.68, 11–29.
Quail, P. H., 1979: Plant cell fractionation. Ann. Rev. Plant Physiol.30, 425–483.
Reid, E., (ed.), 1979: Plant Organelles. New York: J. Wiley and Sons.
Roland, J. C., Lembi, C. A., Morré, D. J., 1972: Phosphotungstic acid as a selective electron-dense stain for plasma membrane of plant cells. Stain Tech.47, 195–200.
Scarborough, G. A., Schulte, T. H., 1974: An improved method for the production ofNeurospora crassa spheroplasts and a new method for measuring transport in fragile cells. Anal. Biochem.61, 441–447.
Sjöstrand, F. S., 1963: A comparison of plasma membrane, cytomembranes, and mitochondrial membranes with respect to ultrastructural features. J. Ultrastruct. Res.9, 561–580.
Twohig, F. M., 1974: Identification of tonoplast fragments in membrane fractions from onion stems. M. S. thesis, Purdue University, West Lafayette, Indiana, U.S.A.
Vander Wilden, W., Matile, P., Schellenberg, M., Meyer, M., Wiemken, A., 1973: Vacuolar membranes: Isolation from yeast cells. Z. Naturforsch.28c, 416–421.
Wehrli, E., Boehm, C., Fuhrman, G. F., 1975: Yeast plasma membrane vesicles suitable for transport studies. J. Bacteriol.124, 1594–1597.
Wiemken, A., Nurse, P., 1973: Isolation and characterization of amino acid pools isolated within the cytoplasm and vacuoles ofCandida utilis. Planta109, 293–306.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Powell, M.J., Bracker, C.E. & Morré, D.J. Isolation and ultrastructural identification of membranes from the fungusGilbertella persicaria . Protoplasma 111, 87–106 (1982). https://doi.org/10.1007/BF01282067
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01282067