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. 1997 Mar;17(3):1093–1101. doi: 10.1128/mcb.17.3.1093

Functional identification of a Leishmania gene related to the peroxin 2 gene reveals common ancestry of glycosomes and peroxisomes.

J A Flaspohler 1, W L Rickoll 1, S M Beverley 1, M Parsons 1
PMCID: PMC231834  PMID: 9032236

Abstract

Glycosomes are membrane-bounded microbody organelles that compartmentalize glycolysis as well as other important metabolic processes in trypanosomatids. The compartmentalization of these enzymatic reactions is hypothesized to play a crucial role in parasite physiology. Although the metabolic role of glycosomes differs substantially from that of the peroxisomes that are found in other eukaryotes, similarities in signals targeting proteins to these organelles suggest that glycosomes and peroxisomes may have evolved from a common ancestor. To examine this hypothesis, as well as gain insights into the function of the glycosome, we used a positive genetic selection procedure to isolate the first Leishmania mutant (gim1-1 [glycosome import] mutant) with a defect in the import of glycosomal proteins. The mutant retains glycosomes but mislocalizes a subset glycosomal proteins to the cytoplasm. Unexpectedly, the gim1-1 mutant lacks lipid bodies, suggesting a heretofore unknown role of the glycosome. We used genetic approaches to identify a gene, GIM1, that is able to restore import and lipid bodies. A nonsense mutation was found in one allele of this gene in the mutant line. The predicted Gim1 protein is related the peroxin 2 family of integral membrane proteins, which are required for peroxisome biogenesis. The similarities in sequence and function provide strong support for the common origin model of glycosomes and peroxisomes. The novel phenotype of gim1-1 and distinctive role of Leishmania glycosomes suggest that future studies of this system will provide a new perspective on microbody biogenesis and function.

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Selected References

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  1. Alexander K., Parsons M. A phosphoglycerate kinase-like molecule localized to glycosomal microbodies: evidence that the topogenic signal is not at the C-terminus. Mol Biochem Parasitol. 1991 May;46(1):1–10. doi: 10.1016/0166-6851(91)90193-a. [DOI] [PubMed] [Google Scholar]
  2. Allen T. E., Hwang H. Y., Jardim A., Olafson R., Ullman B. Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani. Mol Biochem Parasitol. 1995 Jul;73(1-2):133–143. doi: 10.1016/0166-6851(94)00105-v. [DOI] [PubMed] [Google Scholar]
  3. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  4. Ashford R. W., Desjeux P., Deraadt P. Estimation of population at risk of infection and number of cases of Leishmaniasis. Parasitol Today. 1992 Mar;8(3):104–105. doi: 10.1016/0169-4758(92)90249-2. [DOI] [PubMed] [Google Scholar]
  5. Berteaux-Lecellier V., Picard M., Thompson-Coffe C., Zickler D., Panvier-Adoutte A., Simonet J. M. A nonmammalian homolog of the PAF1 gene (Zellweger syndrome) discovered as a gene involved in caryogamy in the fungus Podospora anserina. Cell. 1995 Jun 30;81(7):1043–1051. doi: 10.1016/s0092-8674(05)80009-1. [DOI] [PubMed] [Google Scholar]
  6. Blattner J., Dörsam H., Clatyon C. E. Function of N-terminal import signals in trypanosome microbodies. FEBS Lett. 1995 Mar 6;360(3):310–314. doi: 10.1016/0014-5793(95)00128-v. [DOI] [PubMed] [Google Scholar]
  7. Blattner J., Swinkels B., Dörsam H., Prospero T., Subramani S., Clayton C. Glycosome assembly in trypanosomes: variations in the acceptable degeneracy of a COOH-terminal microbody targeting signal. J Cell Biol. 1992 Dec;119(5):1129–1136. doi: 10.1083/jcb.119.5.1129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Borst P. Peroxisome biogenesis revisited. Biochim Biophys Acta. 1989 Jun 1;1008(1):1–13. doi: 10.1016/0167-4781(89)90163-2. [DOI] [PubMed] [Google Scholar]
  9. Court D. A., Kleene R., Neupert W., Lill R. Role of the N- and C-termini of porin in import into the outer membrane of Neurospora mitochondria. FEBS Lett. 1996 Jul 15;390(1):73–77. doi: 10.1016/0014-5793(96)00629-1. [DOI] [PubMed] [Google Scholar]
  10. Cruz A., Coburn C. M., Beverley S. M. Double targeted gene replacement for creating null mutants. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7170–7174. doi: 10.1073/pnas.88.16.7170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Distel B., Erdmann R., Gould S. J., Blobel G., Crane D. I., Cregg J. M., Dodt G., Fujiki Y., Goodman J. M., Just W. W. A unified nomenclature for peroxisome biogenesis factors. J Cell Biol. 1996 Oct;135(1):1–3. doi: 10.1083/jcb.135.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Elgersma Y., van den Berg M., Tabak H. F., Distel B. An efficient positive selection procedure for the isolation of peroxisomal import and peroxisome assembly mutants of Saccharomyces cerevisiae. Genetics. 1993 Nov;135(3):731–740. doi: 10.1093/genetics/135.3.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Erdmann R., Veenhuis M., Mertens D., Kunau W. H. Isolation of peroxisome-deficient mutants of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5419–5423. doi: 10.1073/pnas.86.14.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Freemont P. S., Hanson I. M., Trowsdale J. A novel cysteine-rich sequence motif. Cell. 1991 Feb 8;64(3):483–484. doi: 10.1016/0092-8674(91)90229-r. [DOI] [PubMed] [Google Scholar]
  15. Gatignol A., Baron M., Tiraby G. Phleomycin resistance encoded by the ble gene from transposon Tn 5 as a dominant selectable marker in Saccharomyces cerevisiae. Mol Gen Genet. 1987 May;207(2-3):342–348. doi: 10.1007/BF00331599. [DOI] [PubMed] [Google Scholar]
  16. Gish W., States D. J. Identification of protein coding regions by database similarity search. Nat Genet. 1993 Mar;3(3):266–272. doi: 10.1038/ng0393-266. [DOI] [PubMed] [Google Scholar]
  17. Gould S. J., Keller G. A., Hosken N., Wilkinson J., Subramani S. A conserved tripeptide sorts proteins to peroxisomes. J Cell Biol. 1989 May;108(5):1657–1664. doi: 10.1083/jcb.108.5.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gould S. J., McCollum D., Spong A. P., Heyman J. A., Subramani S. Development of the yeast Pichia pastoris as a model organism for a genetic and molecular analysis of peroxisome assembly. Yeast. 1992 Aug;8(8):613–628. doi: 10.1002/yea.320080805. [DOI] [PubMed] [Google Scholar]
  19. Hajra A. K. Glycerolipid biosynthesis in peroxisomes (microbodies). Prog Lipid Res. 1995;34(4):343–364. doi: 10.1016/0163-7827(95)00013-5. [DOI] [PubMed] [Google Scholar]
  20. Hannaert V., Blaauw M., Kohl L., Allert S., Opperdoes F. R., Michels P. A. Molecular analysis of the cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase in Leishmania mexicana. Mol Biochem Parasitol. 1992 Oct;55(1-2):115–126. doi: 10.1016/0166-6851(92)90132-4. [DOI] [PubMed] [Google Scholar]
  21. Hart D. T., Opperdoes F. R. The occurrence of glycosomes (microbodies) in the promastigote stage of four major Leishmania species. Mol Biochem Parasitol. 1984 Oct;13(2):159–172. doi: 10.1016/0166-6851(84)90110-5. [DOI] [PubMed] [Google Scholar]
  22. Henikoff S. Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 1987;155:156–165. doi: 10.1016/0076-6879(87)55014-5. [DOI] [PubMed] [Google Scholar]
  23. Heymans H. S., Schutgens R. B., Tan R., van den Bosch H., Borst P. Severe plasmalogen deficiency in tissues of infants without peroxisomes (Zellweger syndrome). Nature. 1983 Nov 3;306(5938):69–70. doi: 10.1038/306069a0. [DOI] [PubMed] [Google Scholar]
  24. Iovannisci D. M., Goebel D., Allen K., Kaur K., Ullman B. Genetic analysis of adenine metabolism in Leishmania donovani promastigotes. Evidence for diploidy at the adenine phosphoribosyltransferase locus. J Biol Chem. 1984 Dec 10;259(23):14617–14623. [PubMed] [Google Scholar]
  25. Iovannisci D. M., Ullman B. Characterization of a mutant Leishmania donovani deficient in adenosine kinase activity. Mol Biochem Parasitol. 1984 Jun;12(2):139–151. doi: 10.1016/0166-6851(84)90131-2. [DOI] [PubMed] [Google Scholar]
  26. Kapler G. M., Coburn C. M., Beverley S. M. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol. 1990 Mar;10(3):1084–1094. doi: 10.1128/mcb.10.3.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. King D. L., Turco S. J. A ricin agglutinin-resistant clone of Leishmania donovani deficient in lipophosphoglycan. Mol Biochem Parasitol. 1988 Apr;28(3):285–293. doi: 10.1016/0166-6851(88)90013-8. [DOI] [PubMed] [Google Scholar]
  28. Kunau W. H., Dommes V., Schulz H. beta-oxidation of fatty acids in mitochondria, peroxisomes, and bacteria: a century of continued progress. Prog Lipid Res. 1995;34(4):267–342. doi: 10.1016/0163-7827(95)00011-9. [DOI] [PubMed] [Google Scholar]
  29. LeBowitz J. H., Coburn C. M., McMahon-Pratt D., Beverley S. M. Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9736–9740. doi: 10.1073/pnas.87.24.9736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mallinson D. J., Coombs G. H. Biochemical characteristics of the metacyclic forms of Leishmania major and L. mexicana mexicana. Parasitology. 1989 Feb;98(Pt 1):7–15. doi: 10.1017/s0031182000059631. [DOI] [PubMed] [Google Scholar]
  31. Michels P. A. Compartmentation of glycolysis in trypanosomes: a potential target for new trypanocidal drugs. Biol Cell. 1988;64(2):157–164. doi: 10.1016/0248-4900(88)90075-5. [DOI] [PubMed] [Google Scholar]
  32. Opperdoes F. R. Localization of the initial steps in alkoxyphospholipid biosynthesis in glycosomes (microbodies) of Trypanosoma brucei. FEBS Lett. 1984 Apr 9;169(1):35–39. doi: 10.1016/0014-5793(84)80284-7. [DOI] [PubMed] [Google Scholar]
  33. Opperdoes F. R., Michels P. A. The glycosomes of the Kinetoplastida. Biochimie. 1993;75(3-4):231–234. doi: 10.1016/0300-9084(93)90081-3. [DOI] [PubMed] [Google Scholar]
  34. Osinga K. A., Swinkels B. W., Gibson W. C., Borst P., Veeneman G. H., Van Boom J. H., Michels P. A., Opperdoes F. R. Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei. EMBO J. 1985 Dec 30;4(13B):3811–3817. doi: 10.1002/j.1460-2075.1985.tb04152.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Parker H. L., Hill T., Alexander K., Murphy N. B., Fish W. R., Parsons M. Three genes and two isozymes: gene conversion and the compartmentalization and expression of the phosphoglycerate kinases of Trypanosoma (Nannomonas) congolense. Mol Biochem Parasitol. 1995 Feb;69(2):269–279. doi: 10.1016/0166-6851(94)00208-5. [DOI] [PubMed] [Google Scholar]
  36. Parsons M., Hill T. Elevated phosphoglycerate kinase mRNA but not protein in monomorphic Trypanosoma brucei: implications for stage-regulation and post-transcriptional control. Mol Biochem Parasitol. 1989 Mar 15;33(3):215–227. doi: 10.1016/0166-6851(89)90083-2. [DOI] [PubMed] [Google Scholar]
  37. Parsons M., Nielsen B. Trypanosoma brucei: two-dimensional gel analysis of the major glycosomal proteins during the life cycle. Exp Parasitol. 1990 Apr;70(3):276–285. doi: 10.1016/0014-4894(90)90109-p. [DOI] [PubMed] [Google Scholar]
  38. Patarca R., Fletcher M. A. Ring finger in the peroxisome assembly factor-1. FEBS Lett. 1992 Nov 2;312(1):1–2. doi: 10.1016/0014-5793(92)81397-5. [DOI] [PubMed] [Google Scholar]
  39. Purdue P. E., Lazarow P. B. Peroxisomal biogenesis: multiple pathways of protein import. J Biol Chem. 1994 Dec 2;269(48):30065–30068. [PubMed] [Google Scholar]
  40. Ryan K. A., Dasgupta S., Beverley S. M. Shuttle cosmid vectors for the trypanosomatid parasite Leishmania. Gene. 1993 Sep 6;131(1):145–150. doi: 10.1016/0378-1119(93)90684-u. [DOI] [PubMed] [Google Scholar]
  41. Sato T. A modified method for lead staining of thin sections. J Electron Microsc (Tokyo) 1968;17(2):158–159. [PubMed] [Google Scholar]
  42. Schulz I. Permeabilizing cells: some methods and applications for the study of intracellular processes. Methods Enzymol. 1990;192:280–300. doi: 10.1016/0076-6879(90)92077-q. [DOI] [PubMed] [Google Scholar]
  43. Shimozawa N., Tsukamoto T., Suzuki Y., Orii T., Shirayoshi Y., Mori T., Fujiki Y. A human gene responsible for Zellweger syndrome that affects peroxisome assembly. Science. 1992 Feb 28;255(5048):1132–1134. doi: 10.1126/science.1546315. [DOI] [PubMed] [Google Scholar]
  44. Skeiky Y. A., Benson D. R., Parsons M., Elkon K. B., Reed S. G. Cloning and expression of Trypanosoma cruzi ribosomal protein P0 and epitope analysis of anti-P0 autoantibodies in Chagas' disease patients. J Exp Med. 1992 Jul 1;176(1):201–211. doi: 10.1084/jem.176.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sommer J. M., Cheng Q. L., Keller G. A., Wang C. C. In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal. Mol Biol Cell. 1992 Jul;3(7):749–759. doi: 10.1091/mbc.3.7.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sommer J. M., Nguyen T. T., Wang C. C. Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C-terminal sequence. FEBS Lett. 1994 Aug 15;350(1):125–129. doi: 10.1016/0014-5793(94)00747-0. [DOI] [PubMed] [Google Scholar]
  47. Sommer J. M., Peterson G., Keller G. A., Parsons M., Wang C. C. The C-terminal tripeptide of glycosomal phosphoglycerate kinase is both necessary and sufficient for import into the glycosomes of Trypanosoma brucei. FEBS Lett. 1993 Jan 18;316(1):53–58. doi: 10.1016/0014-5793(93)81735-i. [DOI] [PubMed] [Google Scholar]
  48. Sommer J. M., Wang C. C. Targeting proteins to the glycosomes of African trypanosomes. Annu Rev Microbiol. 1994;48:105–138. doi: 10.1146/annurev.mi.48.100194.000541. [DOI] [PubMed] [Google Scholar]
  49. Swinkels B. W., Loiseau A., Opperdoes F. R., Borst P. A phosphoglycerate kinase-related gene conserved between Trypanosoma brucei and Crithidia fasciculata. Mol Biochem Parasitol. 1992 Jan;50(1):69–78. doi: 10.1016/0166-6851(92)90245-f. [DOI] [PubMed] [Google Scholar]
  50. Szilard R. K., Titorenko V. I., Veenhuis M., Rachubinski R. A. Pay32p of the yeast Yarrowia lipolytica is an intraperoxisomal component of the matrix protein translocation machinery. J Cell Biol. 1995 Dec;131(6 Pt 1):1453–1469. doi: 10.1083/jcb.131.6.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tan X., Titorenko V. I., van der Klei I. J., Sulter G. J., Haima P., Waterham H. R., Eyers M., Harder W., Veenhuis M., Cregg J. M. Characterization of peroxisome-deficient mutants of Hansenula polymorpha. Curr Genet. 1995 Aug;28(3):248–257. doi: 10.1007/BF00309784. [DOI] [PubMed] [Google Scholar]
  52. Tsukamoto T., Miura S., Fujiki Y. Restoration by a 35K membrane protein of peroxisome assembly in a peroxisome-deficient mammalian cell mutant. Nature. 1991 Mar 7;350(6313):77–81. doi: 10.1038/350077a0. [DOI] [PubMed] [Google Scholar]
  53. Tsukamoto T., Shimozawa N., Fujiki Y. Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis. Mol Cell Biol. 1994 Aug;14(8):5458–5465. doi: 10.1128/mcb.14.8.5458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Turco S. J., Descoteaux A. The lipophosphoglycan of Leishmania parasites. Annu Rev Microbiol. 1992;46:65–94. doi: 10.1146/annurev.mi.46.100192.000433. [DOI] [PubMed] [Google Scholar]
  55. Van der Leij I., Van den Berg M., Boot R., Franse M., Distel B., Tabak H. F. Isolation of peroxisome assembly mutants from Saccharomyces cerevisiae with different morphologies using a novel positive selection procedure. J Cell Biol. 1992 Oct;119(1):153–162. doi: 10.1083/jcb.119.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Waterham H. R., de Vries Y., Russel K. A., Xie W., Veenhuis M., Cregg J. M. The Pichia pastoris PER6 gene product is a peroxisomal integral membrane protein essential for peroxisome biogenesis and has sequence similarity to the Zellweger syndrome protein PAF-1. Mol Cell Biol. 1996 May;16(5):2527–2536. doi: 10.1128/mcb.16.5.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]

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