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. 1982 Aug;151(2):648–656. doi: 10.1128/jb.151.2.648-656.1982

Isolation and characterization of plasma membranes from the fungus Podospora anserina.

J Labarère, M Bonneu
PMCID: PMC220305  PMID: 6212578

Abstract

This paper describes a method for separating and isolating plasma membranes from the septated fungus Podospora anserina. Plasma membranes were isolated from protoplasts (young cell plasma membranes) and mycelia (both young and aged cell plasma membranes). The procedure of fractionation consisted of a combination of differential and isopycnic centrifugations. Characterization of cellular membranes and enrichment of the fractions with plasmalemma were carried out by assays on enzymatic activities. A plasma membrane fraction was isolated in a buoyant density peak of 1.087 g/cm3, where three enzymatic activities bound to plasma membrane, adenylate cyclase, chitin synthase, and beta-glucan synthase at low affinity for UDP-Glc, peaked together. Good purity of this fraction was determined by the absence or the very low level of other enzymatic activities used as markers for intracellular membranes, i.e., succinate dehydrogenase, alpha-mannosidase, NADPH cytochrome c reductase, and beta-glucan synthase at high affinity for UDP-Glc activities.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowman B. J., Mainzer S. E., Allen K. E., Slayman C. W. Effects of inhibitors on the plasma membrane and mitochondrial adenosine triphosphatases of Neurospora crassa. Biochim Biophys Acta. 1978 Sep 11;512(1):13–28. doi: 10.1016/0005-2736(78)90214-6. [DOI] [PubMed] [Google Scholar]
  2. Bowman B. J., Slayman C. W. Characterization of plasma membrane adenosine triphosphatase of Neurospora crassa. J Biol Chem. 1977 May 25;252(10):3357–3363. [PubMed] [Google Scholar]
  3. Bär H. P., Hechter O. Adenyl cyclase and hormone action. I. Effects of adrenocorticotropic hormone, glucagon, and epinephrine on the plasma membrane of rat fat cells. Proc Natl Acad Sci U S A. 1969 Jun;63(2):350–356. doi: 10.1073/pnas.63.2.350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DAVOREN P. R., SUTHERLAND E. W. THE CELLULAR LOCATION OF ADENYL CYCLASE IN THE PIGEON ERYTHROCYTE. J Biol Chem. 1963 Sep;238:3016–3023. [PubMed] [Google Scholar]
  5. Dubé J., Setterfield G., Kiss G., Lusena C. V. Fate of the plasma membrane of Saccharomyces cerevisiae during cell rupture. Can J Microbiol. 1973 Feb;19(2):285–290. doi: 10.1139/m73-043. [DOI] [PubMed] [Google Scholar]
  6. Durrens P., Laigret F., Labarère J., Bernet J. Podospora anserina mutant defective in protoperithecium formation, ascospore germination, and cell regeneration. J Bacteriol. 1979 Dec;140(3):835–842. doi: 10.1128/jb.140.3.835-842.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Durán A., Bowers B., Cabib E. Chitin synthetase zymogen is attached to the yeast plasma membrane. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3952–3955. doi: 10.1073/pnas.72.10.3952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Flawiá M. M., Torres H. N. Adenylate cyclase activity in Neurospora crassa. I. General properties. J Biol Chem. 1972 Nov 10;247(21):6873–6879. [PubMed] [Google Scholar]
  9. Hardin J. W., Cherry J. H., Morré D. J., Lembi C. A. Enhancement of RNA polymerase activity by a factor released by auxin from plasma membrane. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3146–3150. doi: 10.1073/pnas.69.11.3146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Juretić D. The effect of phosphatidylcholine depletion on biochemical and physical properties of a Neurospora crassa membrane mutant. Biochim Biophys Acta. 1977 Sep 5;469(2):137–150. doi: 10.1016/0005-2736(77)90176-6. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Labarere J., Bernet J. Protoplasmic Incompatibility and Cell Lysis in PODOSPORA ANSERINA. I. Genetic Investigations on Mutations of a Novel Modifier Gene That Suppresses Cell Destruction. Genetics. 1977 Oct;87(2):249–257. doi: 10.1093/genetics/87.2.249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Labarere J., Bernet J. Protoplasmic Incompatibility in PODOSPORA ANSERINA: A Possible Role for Its Associated Proteolytic Activity. Genetics. 1979 Nov;93(3):525–537. doi: 10.1093/genetics/93.3.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Longley R. P., Rose A. H., Knights B. A. Composition of the protoplast membrane from Saccharomyces cerevisiae. Biochem J. 1968 Jul;108(3):401–412. doi: 10.1042/bj1080401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lord J. M., Kagawa T., Moore T. S., Beevers H. Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J Cell Biol. 1973 Jun;57(3):659–667. doi: 10.1083/jcb.57.3.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Marriott M. S. Isolation and chemical characterization of plasma membranes from the yeast and mycelial forms of Candida albicans. J Gen Microbiol. 1975 Jan;86(1):115–132. doi: 10.1099/00221287-86-1-115. [DOI] [PubMed] [Google Scholar]
  17. Matile P., Moor H., Mühlethaler K. Isolation and properties of the plasmalemma in yeast. Arch Mikrobiol. 1967;58(3):201–211. doi: 10.1007/BF00408804. [DOI] [PubMed] [Google Scholar]
  18. Mendoza C. G., Villanueva J. R. Preparation and composition of the protoplast membrane of Candida utilis. Biochim Biophys Acta. 1967 May 2;135(2):189–195. doi: 10.1016/0005-2736(67)90113-7. [DOI] [PubMed] [Google Scholar]
  19. Nombela C., Uruburu F., Villanueva J. R. Studies on membranes isolated from extracts of Fusarium culmorum. J Gen Microbiol. 1974 Mar;81(1):247–254. doi: 10.1099/00221287-81-1-247. [DOI] [PubMed] [Google Scholar]
  20. Nurminen T., Oura E., Suomalainen H. The enzymic composition of the isolated cell wall and plasma membrane of baker's yeast. Biochem J. 1970 Jan;116(1):61–69. doi: 10.1042/bj1160061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pohl S. L., Birnbaumer L., Rodbell M. Glucagon-sensitive adenyl cylase in plasma membrane of hepatic parenchymal cells. Science. 1969 May 2;164(3879):566–567. doi: 10.1126/science.164.3879.566. [DOI] [PubMed] [Google Scholar]
  22. Ray P. M., Shininger T. L., Ray M. M. ISOLATION OF beta-GLUCAN SYNTHETASE PARTICLES FROM PLANT CELLS AND IDENTIFICATION WITH GOLGI MEMBRANES. Proc Natl Acad Sci U S A. 1969 Oct;64(2):605–612. doi: 10.1073/pnas.64.2.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rodbell M. Metabolism of isolated fat cells. V. Preparation of "ghosts" and their properties; adenyl cyclase and other enzymes. J Biol Chem. 1967 Dec 25;242(24):5744–5750. [PubMed] [Google Scholar]
  24. SCHATZ G., KLIMA J. TRIPHOSPHOPYRIDINE NUCLEOTIDE: CYTOCHROME C REDUCTASE OF SACCHAROMYCES CEREVISIAE: A "MICROSOMAL" ENZYME. Biochim Biophys Acta. 1964 Mar 9;81:448–461. doi: 10.1016/0926-6569(64)90130-0. [DOI] [PubMed] [Google Scholar]
  25. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  26. Scarborough G. A. Isolation and characterization of Neurospora crassa plasma membranes. J Biol Chem. 1975 Feb 10;250(3):1106–1111. [PubMed] [Google Scholar]
  27. Scarborough G. A. Properties of Neurospora crassa plasma membrane ATPase. Arch Biochem Biophys. 1977 Apr 30;180(2):384–393. doi: 10.1016/0003-9861(77)90052-2. [DOI] [PubMed] [Google Scholar]
  28. Scarborough G. A. The neurospora plasma membrane: a new experimental system for investigating eukaryote surface membrane structure and function. Methods Cell Biol. 1978;20:117–133. doi: 10.1016/s0091-679x(08)62014-2. [DOI] [PubMed] [Google Scholar]
  29. Schibeci A., Rattray J. B., Kidby D. K. Isolation and identification of yeast plasma membrane. Biochim Biophys Acta. 1973 Jun 7;311(1):15–25. doi: 10.1016/0005-2736(73)90250-2. [DOI] [PubMed] [Google Scholar]
  30. Schnaitman C., Erwin V. G., Greenawalt J. W. The submitochondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J Cell Biol. 1967 Mar;32(3):719–735. doi: 10.1083/jcb.32.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Scott W. A. Adenosine 3':5'-cyclic monophosphate deficiency in Neurospora crassa. Proc Natl Acad Sci U S A. 1976 Sep;73(9):2995–2999. doi: 10.1073/pnas.73.9.2995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Serrano R. Characterization of the plasma membrane ATPase of Saccharomyces cerevisiae. Mol Cell Biochem. 1978 Nov 30;22(1):51–63. doi: 10.1007/BF00241470. [DOI] [PubMed] [Google Scholar]
  33. Stroobant P., Scarborough G. A. Large scale isolation and storage of Neurospora plasma membranes. Anal Biochem. 1979 Jun;95(2):554–558. doi: 10.1016/0003-2697(79)90771-1. [DOI] [PubMed] [Google Scholar]
  34. Van Der Woude W. J., Lembi C. A., Morré D. J. beta-Glucan Synthetases of Plasma Membrane and Golgi Apparatus from Onion Stem. Plant Physiol. 1974 Sep;54(3):333–340. doi: 10.1104/pp.54.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]

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