Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Oct 1;131(1):19–31. doi: 10.1083/jcb.131.1.19

Isolation and characterization of nuclear envelopes from the yeast Saccharomyces

PMCID: PMC2120596  PMID: 7559775

Abstract

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.

Full Text

The Full Text of this article is available as a PDF (3.1 MB).

Selected References

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

  1. Adam E. J., Adam S. A. Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope. J Cell Biol. 1994 May;125(3):547–555. doi: 10.1083/jcb.125.3.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akey C. W., Radermacher M. Architecture of the Xenopus nuclear pore complex revealed by three-dimensional cryo-electron microscopy. J Cell Biol. 1993 Jul;122(1):1–19. doi: 10.1083/jcb.122.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Black S. D., Coon M. J. Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region. J Biol Chem. 1982 May 25;257(10):5929–5938. [PubMed] [Google Scholar]
  4. Brodsky J. L., Hamamoto S., Feldheim D., Schekman R. Reconstitution of protein translocation from solubilized yeast membranes reveals topologically distinct roles for BiP and cytosolic Hsc70. J Cell Biol. 1993 Jan;120(1):95–102. doi: 10.1083/jcb.120.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chirico W. J., Waters M. G., Blobel G. 70K heat shock related proteins stimulate protein translocation into microsomes. Nature. 1988 Apr 28;332(6167):805–810. doi: 10.1038/332805a0. [DOI] [PubMed] [Google Scholar]
  6. Courvalin J. C., Dumontier M., Bornens M. Solubilization of nuclear structures by the polyanion heparin. J Biol Chem. 1982 Jan 10;257(1):456–463. [PubMed] [Google Scholar]
  7. Cox J. S., Shamu C. E., Walter P. Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 1993 Jun 18;73(6):1197–1206. doi: 10.1016/0092-8674(93)90648-a. [DOI] [PubMed] [Google Scholar]
  8. Davis L. I., Fink G. R. The NUP1 gene encodes an essential component of the yeast nuclear pore complex. Cell. 1990 Jun 15;61(6):965–978. doi: 10.1016/0092-8674(90)90062-j. [DOI] [PubMed] [Google Scholar]
  9. Dresser M. E., Giroux C. N. Meiotic chromosome behavior in spread preparations of yeast. J Cell Biol. 1988 Mar;106(3):567–573. doi: 10.1083/jcb.106.3.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fried H. M., Warner J. R. Cloning of yeast gene for trichodermin resistance and ribosomal protein L3. Proc Natl Acad Sci U S A. 1981 Jan;78(1):238–242. doi: 10.1073/pnas.78.1.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Georgatos S. D., Maroulakou I., Blobel G. Lamin A, lamin B, and lamin B receptor analogues in yeast. J Cell Biol. 1989 Jun;108(6):2069–2082. doi: 10.1083/jcb.108.6.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gilson E., Laroche T., Gasser S. M. Telomeres and the functional architecture of the nucleus. Trends Cell Biol. 1993 Apr;3(4):128–134. doi: 10.1016/0962-8924(93)90175-z. [DOI] [PubMed] [Google Scholar]
  14. Grandi P., Schlaich N., Tekotte H., Hurt E. C. Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p. EMBO J. 1995 Jan 3;14(1):76–87. doi: 10.1002/j.1460-2075.1995.tb06977.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Görlich D., Prehn S., Laskey R. A., Hartmann E. Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 1994 Dec 2;79(5):767–778. doi: 10.1016/0092-8674(94)90067-1. [DOI] [PubMed] [Google Scholar]
  16. Hansen W., Garcia P. D., Walter P. In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor. Cell. 1986 May 9;45(3):397–406. doi: 10.1016/0092-8674(86)90325-9. [DOI] [PubMed] [Google Scholar]
  17. Hardwick K. G., Pelham H. R. SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J Cell Biol. 1992 Nov;119(3):513–521. doi: 10.1083/jcb.119.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hurt E. C., Mutvei A., Carmo-Fonseca M. The nuclear envelope of the yeast Saccharomyces cerevisiae. Int Rev Cytol. 1992;136:145–184. doi: 10.1016/s0074-7696(08)62052-5. [DOI] [PubMed] [Google Scholar]
  19. Kilmartin J. V., Adams A. E. Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces. J Cell Biol. 1984 Mar;98(3):922–933. doi: 10.1083/jcb.98.3.922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kilmartin J. V., Dyos S. L., Kershaw D., Finch J. T. A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated. J Cell Biol. 1993 Dec;123(5):1175–1184. doi: 10.1083/jcb.123.5.1175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kilmartin J. V. Genetic and biochemical approaches to spindle function and chromosome segregation in eukaryotic microorganisms. Curr Opin Cell Biol. 1994 Feb;6(1):50–54. doi: 10.1016/0955-0674(94)90115-5. [DOI] [PubMed] [Google Scholar]
  22. Klein F., Laroche T., Cardenas M. E., Hofmann J. F., Schweizer D., Gasser S. M. Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast. J Cell Biol. 1992 Jun;117(5):935–948. doi: 10.1083/jcb.117.5.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Latterich M., Schekman R. The karyogamy gene KAR2 and novel proteins are required for ER-membrane fusion. Cell. 1994 Jul 15;78(1):87–98. doi: 10.1016/0092-8674(94)90575-4. [DOI] [PubMed] [Google Scholar]
  24. Loeb J. D., Davis L. I., Fink G. R. NUP2, a novel yeast nucleoporin, has functional overlap with other proteins of the nuclear pore complex. Mol Biol Cell. 1993 Feb;4(2):209–222. doi: 10.1091/mbc.4.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mann K., Mecke D. The Triton X-100 and high salt resistant residue of Saccharomyces cerevisiae nuclear membranes. Z Naturforsch C. 1982 Oct;37(10):916–920. doi: 10.1515/znc-1982-1011. [DOI] [PubMed] [Google Scholar]
  26. Mann K., Mecke D. The isolation of Saccharomyces cerevisiae nuclear membranes with nuclease and high-salt treatment. Biochim Biophys Acta. 1982 Apr 23;687(1):57–62. doi: 10.1016/0005-2736(82)90169-9. [DOI] [PubMed] [Google Scholar]
  27. Masuda H., Sevik M., Cande W. Z. In vitro microtubule-nucleating activity of spindle pole bodies in fission yeast Schizosaccharomyces pombe: cell cycle-dependent activation in xenopus cell-free extracts. J Cell Biol. 1992 Jun;117(5):1055–1066. doi: 10.1083/jcb.117.5.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Moore M. S., Blobel G. A G protein involved in nucleocytoplasmic transport: the role of Ran. Trends Biochem Sci. 1994 May;19(5):211–216. doi: 10.1016/0968-0004(94)90024-8. [DOI] [PubMed] [Google Scholar]
  29. Moore M. S., Blobel G. Purification of a Ran-interacting protein that is required for protein import into the nucleus. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10212–10216. doi: 10.1073/pnas.91.21.10212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moore M. S., Blobel G. The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature. 1993 Oct 14;365(6447):661–663. doi: 10.1038/365661a0. [DOI] [PubMed] [Google Scholar]
  31. Mori K., Ma W., Gething M. J., Sambrook J. A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 1993 Aug 27;74(4):743–756. doi: 10.1016/0092-8674(93)90521-q. [DOI] [PubMed] [Google Scholar]
  32. Mortimer R. K., Johnston J. R. Genealogy of principal strains of the yeast genetic stock center. Genetics. 1986 May;113(1):35–43. doi: 10.1093/genetics/113.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mutvei A., Dihlmann S., Herth W., Hurt E. C. NSP1 depletion in yeast affects nuclear pore formation and nuclear accumulation. Eur J Cell Biol. 1992 Dec;59(2):280–295. [PubMed] [Google Scholar]
  34. Napier R. M., Fowke L. C., Hawes C., Lewis M., Pelham H. R. Immunological evidence that plants use both HDEL and KDEL for targeting proteins to the endoplasmic reticulum. J Cell Sci. 1992 Jun;102(Pt 2):261–271. doi: 10.1242/jcs.102.2.261. [DOI] [PubMed] [Google Scholar]
  35. Nehrbass U., Kern H., Mutvei A., Horstmann H., Marshallsay B., Hurt E. C. NSP1: a yeast nuclear envelope protein localized at the nuclear pores exerts its essential function by its carboxy-terminal domain. Cell. 1990 Jun 15;61(6):979–989. doi: 10.1016/0092-8674(90)90063-k. [DOI] [PubMed] [Google Scholar]
  36. Normington K., Kohno K., Kozutsumi Y., Gething M. J., Sambrook J. S. cerevisiae encodes an essential protein homologous in sequence and function to mammalian BiP. Cell. 1989 Jun 30;57(7):1223–1236. doi: 10.1016/0092-8674(89)90059-7. [DOI] [PubMed] [Google Scholar]
  37. Palladino F., Laroche T., Gilson E., Axelrod A., Pillus L., Gasser S. M. SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres. Cell. 1993 Nov 5;75(3):543–555. doi: 10.1016/0092-8674(93)90388-7. [DOI] [PubMed] [Google Scholar]
  38. Preuss D., Mulholland J., Kaiser C. A., Orlean P., Albright C., Rose M. D., Robbins P. W., Botstein D. Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy. Yeast. 1991 Dec;7(9):891–911. doi: 10.1002/yea.320070902. [DOI] [PubMed] [Google Scholar]
  39. Radu A., Blobel G., Moore M. S. Identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1769–1773. doi: 10.1073/pnas.92.5.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rexach M. F., Latterich M., Schekman R. W. Characteristics of endoplasmic reticulum-derived transport vesicles. J Cell Biol. 1994 Sep;126(5):1133–1148. doi: 10.1083/jcb.126.5.1133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rose M. D., Misra L. M., Vogel J. P. KAR2, a karyogamy gene, is the yeast homolog of the mammalian BiP/GRP78 gene. Cell. 1989 Jun 30;57(7):1211–1221. doi: 10.1016/0092-8674(89)90058-5. [DOI] [PubMed] [Google Scholar]
  42. Rothblatt J. A., Deshaies R. J., Sanders S. L., Daum G., Schekman R. Multiple genes are required for proper insertion of secretory proteins into the endoplasmic reticulum in yeast. J Cell Biol. 1989 Dec;109(6 Pt 1):2641–2652. doi: 10.1083/jcb.109.6.2641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rothblatt J. A., Meyer D. I. Secretion in yeast: reconstitution of the translocation and glycosylation of alpha-factor and invertase in a homologous cell-free system. Cell. 1986 Feb 28;44(4):619–628. doi: 10.1016/0092-8674(86)90271-0. [DOI] [PubMed] [Google Scholar]
  44. Rout M. P., Blobel G. Isolation of the yeast nuclear pore complex. J Cell Biol. 1993 Nov;123(4):771–783. doi: 10.1083/jcb.123.4.771. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rout M. P., Kilmartin J. V. Components of the yeast spindle and spindle pole body. J Cell Biol. 1990 Nov;111(5 Pt 1):1913–1927. doi: 10.1083/jcb.111.5.1913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Rout M. P., Kilmartin J. V. Yeast spindle pole body components. Cold Spring Harb Symp Quant Biol. 1991;56:687–692. doi: 10.1101/sqb.1991.056.01.077. [DOI] [PubMed] [Google Scholar]
  47. Rout M. P., Wente S. R. Pores for thought: nuclear pore complex proteins. Trends Cell Biol. 1994 Oct;4(10):357–365. doi: 10.1016/0962-8924(94)90085-x. [DOI] [PubMed] [Google Scholar]
  48. Stirling C. J., Rothblatt J., Hosobuchi M., Deshaies R., Schekman R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. Mol Biol Cell. 1992 Feb;3(2):129–142. doi: 10.1091/mbc.3.2.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sutter T. R., Loper J. C. Disruption of the Saccharomyces cerevisiae gene for NADPH-cytochrome P450 reductase causes increased sensitivity to ketoconazole. Biochem Biophys Res Commun. 1989 May 15;160(3):1257–1266. doi: 10.1016/s0006-291x(89)80139-1. [DOI] [PubMed] [Google Scholar]
  50. Søgaard M., Tani K., Ye R. R., Geromanos S., Tempst P., Kirchhausen T., Rothman J. E., Söllner T. A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell. 1994 Sep 23;78(6):937–948. doi: 10.1016/0092-8674(94)90270-4. [DOI] [PubMed] [Google Scholar]
  51. Waters M. G., Blobel G. Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis. J Cell Biol. 1986 May;102(5):1543–1550. doi: 10.1083/jcb.102.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Waters M. G., Chirico W. J., Blobel G. Protein translocation across the yeast microsomal membrane is stimulated by a soluble factor. J Cell Biol. 1986 Dec;103(6 Pt 2):2629–2636. doi: 10.1083/jcb.103.6.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Waters M. G., Evans E. A., Blobel G. Prepro-alpha-factor has a cleavable signal sequence. J Biol Chem. 1988 May 5;263(13):6209–6214. [PubMed] [Google Scholar]
  54. Wente S. R., Blobel G. NUP145 encodes a novel yeast glycine-leucine-phenylalanine-glycine (GLFG) nucleoporin required for nuclear envelope structure. J Cell Biol. 1994 Jun;125(5):955–969. doi: 10.1083/jcb.125.5.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wente S. R., Rout M. P., Blobel G. A new family of yeast nuclear pore complex proteins. J Cell Biol. 1992 Nov;119(4):705–723. doi: 10.1083/jcb.119.4.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wozniak R. W., Blobel G., Rout M. P. POM152 is an integral protein of the pore membrane domain of the yeast nuclear envelope. J Cell Biol. 1994 Apr;125(1):31–42. doi: 10.1083/jcb.125.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Yabusaki Y., Murakami H., Ohkawa H. Primary structure of Saccharomyces cerevisiae NADPH-cytochrome P450 reductase deduced from nucleotide sequence of its cloned gene. J Biochem. 1988 Jun;103(6):1004–1010. doi: 10.1093/oxfordjournals.jbchem.a122370. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES