Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2004 Jun 15;380(Pt 3):907–918. doi: 10.1042/BJ20031949

Acyl-CoA-binding protein, Acb1p, is required for normal vacuole function and ceramide synthesis in Saccharomyces cerevisiae.

Nils J Faergeman 1, Søren Feddersen 1, Janne K Christiansen 1, Morten K Larsen 1, Roger Schneiter 1, Christian Ungermann 1, Kudzai Mutenda 1, Peter Roepstorff 1, Jens Knudsen 1
PMCID: PMC1224232  PMID: 15032750

Abstract

In the present study, we show that depletion of acyl-CoA-binding protein, Acb1p, in yeast affects ceramide levels, protein trafficking, vacuole fusion and structure. Vacuoles in Acb1p-depleted cells are multi-lobed, contain significantly less of the SNAREs (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptors) Nyv1p, Vam3p and Vti1p, and are unable to fuse in vitro. Mass spectrometric analysis revealed a dramatic reduction in the content of ceramides in whole-cell lipids and in vacuoles isolated from Acb1p-depleted cells. Maturation of yeast aminopeptidase I and carboxypeptidase Y is slightly delayed in Acb1p-depleted cells, whereas the maturation of alkaline phosphatase and Gas1p is unaffected. The fact that Gas1p maturation is unaffected by Acb1p depletion, despite the lowered ceramide content in these cells, indicates that ceramide synthesis in yeast could be compartmentalized. We suggest that the reduced ceramide synthesis in Acb1p-depleted cells leads to severely altered vacuole morphology, perturbed vacuole assembly and strong inhibition of homotypic vacuole fusion.

Full Text

The Full Text of this article is available as a PDF (938.2 KB).

Selected References

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

  1. Albert S., Gallwitz D. Two new members of a family of Ypt/Rab GTPase activating proteins. Promiscuity of substrate recognition. J Biol Chem. 1999 Nov 19;274(47):33186–33189. doi: 10.1074/jbc.274.47.33186. [DOI] [PubMed] [Google Scholar]
  2. Bagnat M., Chang A., Simons K. Plasma membrane proton ATPase Pma1p requires raft association for surface delivery in yeast. Mol Biol Cell. 2001 Dec;12(12):4129–4138. doi: 10.1091/mbc.12.12.4129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barlowe C. COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles. FEBS Lett. 1995 Aug 1;369(1):93–96. doi: 10.1016/0014-5793(95)00618-j. [DOI] [PubMed] [Google Scholar]
  4. Bryant N. J., Stevens T. H. Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole. Microbiol Mol Biol Rev. 1998 Mar;62(1):230–247. doi: 10.1128/mmbr.62.1.230-247.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burz D. S., Rivera-Pomar R., Jäckle H., Hanes S. D. Cooperative DNA-binding by Bicoid provides a mechanism for threshold-dependent gene activation in the Drosophila embryo. EMBO J. 1998 Oct 15;17(20):5998–6009. doi: 10.1093/emboj/17.20.5998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung Ji-Hyun, Lester Robert L., Dickson Robert C. Sphingolipid requirement for generation of a functional v1 component of the vacuolar ATPase. J Biol Chem. 2003 May 13;278(31):28872–28881. doi: 10.1074/jbc.M300943200. [DOI] [PubMed] [Google Scholar]
  7. Conzelmann A., Riezman H., Desponds C., Bron C. A major 125-kd membrane glycoprotein of Saccharomyces cerevisiae is attached to the lipid bilayer through an inositol-containing phospholipid. EMBO J. 1988 Jul;7(7):2233–2240. doi: 10.1002/j.1460-2075.1988.tb03063.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cook N. R., Davidson H. W. In vitro assays of vesicular transport. Traffic. 2001 Jan;2(1):19–25. doi: 10.1034/j.1600-0854.2001.020104.x. [DOI] [PubMed] [Google Scholar]
  9. Cowles C. R., Odorizzi G., Payne G. S., Emr S. D. The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole. Cell. 1997 Oct 3;91(1):109–118. doi: 10.1016/s0092-8674(01)80013-1. [DOI] [PubMed] [Google Scholar]
  10. Darsow T., Rieder S. E., Emr S. D. A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole. J Cell Biol. 1997 Aug 11;138(3):517–529. doi: 10.1083/jcb.138.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dupré Sophie, Haguenauer-Tsapis Rosine. Raft partitioning of the yeast uracil permease during trafficking along the endocytic pathway. Traffic. 2003 Feb;4(2):83–96. doi: 10.1034/j.1600-0854.2003.40204.x. [DOI] [PubMed] [Google Scholar]
  12. Faergeman N. J., Ballegaard T., Knudsen J., Black P. N., DiRusso C. Possible roles of long-chain fatty Acyl-CoA esters in the fusion of biomembranes. Subcell Biochem. 2000;34:175–231. doi: 10.1007/0-306-46824-7_5. [DOI] [PubMed] [Google Scholar]
  13. Faergeman N. J., Knudsen J. Role of long-chain fatty acyl-CoA esters in the regulation of metabolism and in cell signalling. Biochem J. 1997 Apr 1;323(Pt 1):1–12. doi: 10.1042/bj3230001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gaigg B., Neergaard T. B., Schneiter R., Hansen J. K., Faergeman N. J., Jensen N. A., Andersen J. R., Friis J., Sandhoff R., Schrøder H. D. Depletion of acyl-coenzyme A-binding protein affects sphingolipid synthesis and causes vesicle accumulation and membrane defects in Saccharomyces cerevisiae. Mol Biol Cell. 2001 Apr;12(4):1147–1160. doi: 10.1091/mbc.12.4.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gietz D., St Jean A., Woods R. A., Schiestl R. H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992 Mar 25;20(6):1425–1425. doi: 10.1093/nar/20.6.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guillas I., Kirchman P. A., Chuard R., Pfefferli M., Jiang J. C., Jazwinski S. M., Conzelmann A. C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p. EMBO J. 2001 Jun 1;20(11):2655–2665. doi: 10.1093/emboj/20.11.2655. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Haas A., Conradt B., Wickner W. G-protein ligands inhibit in vitro reactions of vacuole inheritance. J Cell Biol. 1994 Jul;126(1):87–97. doi: 10.1083/jcb.126.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Haas A., Wickner W. Homotypic vacuole fusion requires Sec17p (yeast alpha-SNAP) and Sec18p (yeast NSF). EMBO J. 1996 Jul 1;15(13):3296–3305. [PMC free article] [PubMed] [Google Scholar]
  19. Hamamatsu S., Shibuya I., Takagi M., Ohta A. Loss of phosphatidylserine synthesis results in aberrant solute sequestration and vacuolar morphology in Saccharomyces cerevisiae. FEBS Lett. 1994 Jul 4;348(1):33–36. doi: 10.1016/0014-5793(94)00576-1. [DOI] [PubMed] [Google Scholar]
  20. Hearn John D., Lester Robert L., Dickson Robert C. The uracil transporter Fur4p associates with lipid rafts. J Biol Chem. 2002 Nov 21;278(6):3679–3686. doi: 10.1074/jbc.M209170200. [DOI] [PubMed] [Google Scholar]
  21. Horvath A., Sütterlin C., Manning-Krieg U., Movva N. R., Riezman H. Ceramide synthesis enhances transport of GPI-anchored proteins to the Golgi apparatus in yeast. EMBO J. 1994 Aug 15;13(16):3687–3695. doi: 10.1002/j.1460-2075.1994.tb06678.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kaiser C. A., Schekman R. Distinct sets of SEC genes govern transport vesicle formation and fusion early in the secretory pathway. Cell. 1990 May 18;61(4):723–733. doi: 10.1016/0092-8674(90)90483-u. [DOI] [PubMed] [Google Scholar]
  23. Kato M., Wickner W. Ergosterol is required for the Sec18/ATP-dependent priming step of homotypic vacuole fusion. EMBO J. 2001 Aug 1;20(15):4035–4040. doi: 10.1093/emboj/20.15.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klionsky D. J., Cueva R., Yaver D. S. Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway. J Cell Biol. 1992 Oct;119(2):287–299. doi: 10.1083/jcb.119.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Knudsen J., Faergeman N. J., Skøtt H., Hummel R., Børsting C., Rose T. M., Andersen J. S., Højrup P., Roepstorff P., Kristiansen K. Yeast acyl-CoA-binding protein: acyl-CoA-binding affinity and effect on intracellular acyl-CoA pool size. Biochem J. 1994 Sep 1;302(Pt 2):479–485. doi: 10.1042/bj3020479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kohlwein S. D., Eder S., Oh C. S., Martin C. E., Gable K., Bacikova D., Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol Cell Biol. 2001 Jan;21(1):109–125. doi: 10.1128/MCB.21.1.109-125.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kragelund B. B., Knudsen J., Poulsen F. M. Acyl-coenzyme A binding protein (ACBP). Biochim Biophys Acta. 1999 Nov 23;1441(2-3):150–161. doi: 10.1016/s1388-1981(99)00151-1. [DOI] [PubMed] [Google Scholar]
  28. Longtine M. S., McKenzie A., 3rd, Demarini D. J., Shah N. G., Wach A., Brachat A., Philippsen P., Pringle J. R. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast. 1998 Jul;14(10):953–961. doi: 10.1002/(SICI)1097-0061(199807)14:10<953::AID-YEA293>3.0.CO;2-U. [DOI] [PubMed] [Google Scholar]
  29. Mayer A., Wickner W., Haas A. Sec18p (NSF)-driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast vacuoles. Cell. 1996 Apr 5;85(1):83–94. doi: 10.1016/s0092-8674(00)81084-3. [DOI] [PubMed] [Google Scholar]
  30. Mayer Andreas. Membrane fusion in eukaryotic cells. Annu Rev Cell Dev Biol. 2002 Apr 2;18:289–314. doi: 10.1146/annurev.cellbio.18.032202.114809. [DOI] [PubMed] [Google Scholar]
  31. Munn A. L., Riezman H. Endocytosis is required for the growth of vacuolar H(+)-ATPase-defective yeast: identification of six new END genes. J Cell Biol. 1994 Oct;127(2):373–386. doi: 10.1083/jcb.127.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nichols B. J., Ungermann C., Pelham H. R., Wickner W. T., Haas A. Homotypic vacuolar fusion mediated by t- and v-SNAREs. Nature. 1997 May 8;387(6629):199–202. doi: 10.1038/387199a0. [DOI] [PubMed] [Google Scholar]
  33. Obeid Lina M., Okamoto Yasuo, Mao Cungui. Yeast sphingolipids: metabolism and biology. Biochim Biophys Acta. 2002 Dec 30;1585(2-3):163–171. doi: 10.1016/s1388-1981(02)00337-2. [DOI] [PubMed] [Google Scholar]
  34. Ostermann J., Orci L., Tani K., Amherdt M., Ravazzola M., Elazar Z., Rothman J. E. Stepwise assembly of functionally active transport vesicles. Cell. 1993 Dec 3;75(5):1015–1025. doi: 10.1016/0092-8674(93)90545-2. [DOI] [PubMed] [Google Scholar]
  35. Pan X., Goldfarb D. S. YEB3/VAC8 encodes a myristylated armadillo protein of the Saccharomyces cerevisiae vacuolar membrane that functions in vacuole fusion and inheritance. J Cell Sci. 1998 Aug;111(Pt 15):2137–2147. doi: 10.1242/jcs.111.15.2137. [DOI] [PubMed] [Google Scholar]
  36. Pfanner N., Glick B. S., Arden S. R., Rothman J. E. Fatty acylation promotes fusion of transport vesicles with Golgi cisternae. J Cell Biol. 1990 Apr;110(4):955–961. doi: 10.1083/jcb.110.4.955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pfanner N., Orci L., Glick B. S., Amherdt M., Arden S. R., Malhotra V., Rothman J. E. Fatty acyl-coenzyme A is required for budding of transport vesicles from Golgi cisternae. Cell. 1989 Oct 6;59(1):95–102. doi: 10.1016/0092-8674(89)90872-6. [DOI] [PubMed] [Google Scholar]
  38. Rasmussen J. T., Faergeman N. J., Kristiansen K., Knudsen J. Acyl-CoA-binding protein (ACBP) can mediate intermembrane acyl-CoA transport and donate acyl-CoA for beta-oxidation and glycerolipid synthesis. Biochem J. 1994 Apr 1;299(Pt 1):165–170. doi: 10.1042/bj2990165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Roth M. G. Lipid regulators of membrane traffic through the Golgi complex. Trends Cell Biol. 1999 May;9(5):174–179. doi: 10.1016/s0962-8924(99)01535-4. [DOI] [PubMed] [Google Scholar]
  40. Rothman J. E., Wieland F. T. Protein sorting by transport vesicles. Science. 1996 Apr 12;272(5259):227–234. doi: 10.1126/science.272.5259.227. [DOI] [PubMed] [Google Scholar]
  41. Sattler T., Mayer A. Cell-free reconstitution of microautophagic vacuole invagination and vesicle formation. J Cell Biol. 2000 Oct 30;151(3):529–538. doi: 10.1083/jcb.151.3.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schmidt A., Wolde M., Thiele C., Fest W., Kratzin H., Podtelejnikov A. V., Witke W., Huttner W. B., Söling H. D. Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid. Nature. 1999 Sep 9;401(6749):133–141. doi: 10.1038/43613. [DOI] [PubMed] [Google Scholar]
  43. Schneiter R., Brügger B., Sandhoff R., Zellnig G., Leber A., Lampl M., Athenstaedt K., Hrastnik C., Eder S., Daum G. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. J Cell Biol. 1999 Aug 23;146(4):741–754. doi: 10.1083/jcb.146.4.741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schorling S., Vallée B., Barz W. P., Riezman H., Oesterhelt D. Lag1p and Lac1p are essential for the Acyl-CoA-dependent ceramide synthase reaction in Saccharomyces cerevisae. Mol Biol Cell. 2001 Nov;12(11):3417–3427. doi: 10.1091/mbc.12.11.3417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sütterlin C., Doering T. L., Schimmöller F., Schröder S., Riezman H. Specific requirements for the ER to Golgi transport of GPI-anchored proteins in yeast. J Cell Sci. 1997 Nov;110(Pt 21):2703–2714. doi: 10.1242/jcs.110.21.2703. [DOI] [PubMed] [Google Scholar]
  46. Ungermann C., Nichols B. J., Pelham H. R., Wickner W. A vacuolar v-t-SNARE complex, the predominant form in vivo and on isolated vacuoles, is disassembled and activated for docking and fusion. J Cell Biol. 1998 Jan 12;140(1):61–69. doi: 10.1083/jcb.140.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Veit M., Laage R., Dietrich L., Wang L., Ungermann C. Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion. EMBO J. 2001 Jun 15;20(12):3145–3155. doi: 10.1093/emboj/20.12.3145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vida T. A., Emr S. D. A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol. 1995 Mar;128(5):779–792. doi: 10.1083/jcb.128.5.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wang Y. X., Catlett N. L., Weisman L. S. Vac8p, a vacuolar protein with armadillo repeats, functions in both vacuole inheritance and protein targeting from the cytoplasm to vacuole. J Cell Biol. 1998 Mar 9;140(5):1063–1074. doi: 10.1083/jcb.140.5.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Weigert R., Silletta M. G., Spanò S., Turacchio G., Cericola C., Colanzi A., Senatore S., Mancini R., Polishchuk E. V., Salmona M. CtBP/BARS induces fission of Golgi membranes by acylating lysophosphatidic acid. Nature. 1999 Nov 25;402(6760):429–433. doi: 10.1038/46587. [DOI] [PubMed] [Google Scholar]
  51. Zinser E., Daum G. Isolation and biochemical characterization of organelles from the yeast, Saccharomyces cerevisiae. Yeast. 1995 May;11(6):493–536. doi: 10.1002/yea.320110602. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES