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. 2001 Jun 1;356(Pt 2):335–344. doi: 10.1042/0264-6021:3560335

Characterization of a differentially expressed protein that shows an unusual localization to intracellular membranes in Leishmania major.

E Knuepfer 1, Y D Stierhof 1, P G McKean 1, D F Smith 1
PMCID: PMC1221843  PMID: 11368759

Abstract

The SHERP genes are found as a tandem pair within the differentially regulated LmcDNA16 locus of Leishmania major. The SHERP gene product (small hydrophilic endoplasmic reticulum-associated protein) is unusual in its small size (6.2 kDa), its acidic pI (4.6) and its exclusive, high-level expression ( approximately 100000 copies per cell) in infective non-replicative parasite stages. No homologues have been found to date. Secondary-structure predictions suggest that SHERP contains an amphiphilic alpha-helix that is presumably involved in protein-protein interactions. SHERP has been localized to the endoplasmic reticulum as well as to the outer mitochondrial membrane in both wild-type and over-expressing parasites. Given the absence of an N-terminal signal sequence, transmembrane-spanning domains or detectable post-translational modifications, it is likely that this hydrophilic molecule is a peripheral membrane protein on the cytosolic face of intracellular membranes. This weak membrane association has been confirmed in cell-fractionation assays, in which SHERP redistributes from the cytoplasmic to the membrane fraction after in vivo cross-linking. SHERP does not appear to be involved in rearrangements of the cytoskeleton or conservation of organelle morphology during parasite differentiation. The role of this novel protein, presumed to be part of a protein complex, in infective parasites that are nutrient-deficient and pre-adapted for intracellular survival in the mammalian host is under investigation.

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Selected References

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

  1. Achleitner G., Gaigg B., Krasser A., Kainersdorfer E., Kohlwein S. D., Perktold A., Zellnig G., Daum G. Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur J Biochem. 1999 Sep;264(2):545–553. doi: 10.1046/j.1432-1327.1999.00658.x. [DOI] [PubMed] [Google Scholar]
  2. Al-Qahtani A., Teilhet M., Mensa-Wilmot K. Species-specificity in endoplasmic reticulum signal peptide utilization revealed by proteins from Trypanosoma brucei and Leishmania. Biochem J. 1998 Apr 15;331(Pt 2):521–529. doi: 10.1042/bj3310521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bangs J. D., Uyetake L., Brickman M. J., Balber A. E., Boothroyd J. C. Molecular cloning and cellular localization of a BiP homologue in Trypanosoma brucei. Divergent ER retention signals in a lower eukaryote. J Cell Sci. 1993 Aug;105(Pt 4):1101–1113. doi: 10.1242/jcs.105.4.1101. [DOI] [PubMed] [Google Scholar]
  4. Baschong W., Stierhof Y. D. Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy. Microsc Res Tech. 1998 Jul 1;42(1):66–79. doi: 10.1002/(SICI)1097-0029(19980701)42:1<66::AID-JEMT8>3.0.CO;2-P. [DOI] [PubMed] [Google Scholar]
  5. Beresten S. F., Stan R., van Brabant A. J., Ye T., Naureckiene S., Ellis N. A. Purification of overexpressed hexahistidine-tagged BLM N431 as oligomeric complexes. Protein Expr Purif. 1999 Nov;17(2):239–248. doi: 10.1006/prep.1999.1135. [DOI] [PubMed] [Google Scholar]
  6. Borgese N., Aggujaro D., Carrera P., Pietrini G., Bassetti M. A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes. J Cell Biol. 1996 Dec;135(6 Pt 1):1501–1513. doi: 10.1083/jcb.135.6.1501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brodin T. N., Heath S., Sacks D. L. Genes selectively expressed in the infectious (metacyclic) stage of Leishmania major promastigotes encode a potential basic-zipper structural motif. Mol Biochem Parasitol. 1992 Jun;52(2):241–250. doi: 10.1016/0166-6851(92)90056-p. [DOI] [PubMed] [Google Scholar]
  8. Burchmore R. J., Hart D. T. Glucose transport in amastigotes and promastigotes of Leishmania mexicana mexicana. Mol Biochem Parasitol. 1995 Oct;74(1):77–86. doi: 10.1016/0166-6851(95)02485-9. [DOI] [PubMed] [Google Scholar]
  9. Burchmore R. J., Landfear S. M. Differential regulation of multiple glucose transporter genes in Leishmania mexicana. J Biol Chem. 1998 Oct 30;273(44):29118–29126. doi: 10.1074/jbc.273.44.29118. [DOI] [PubMed] [Google Scholar]
  10. Coulson R. M., Connor V., Ajioka J. W. Using 3' untranslated sequences to identify differentially expressed genes in Leishmania. Gene. 1997 Sep 1;196(1-2):159–164. doi: 10.1016/s0378-1119(97)00221-7. [DOI] [PubMed] [Google Scholar]
  11. Coulson R. M., Connor V., Chen J. C., Ajioka J. W. Differential expression of Leishmania major beta-tubulin genes during the acquisition of promastigote infectivity. Mol Biochem Parasitol. 1996 Nov 25;82(2):227–236. doi: 10.1016/0166-6851(96)02739-9. [DOI] [PubMed] [Google Scholar]
  12. Coulson R. M., Smith D. F. Isolation of genes showing increased or unique expression in the infective promastigotes of Leishmania major. Mol Biochem Parasitol. 1990 Apr;40(1):63–75. doi: 10.1016/0166-6851(90)90080-6. [DOI] [PubMed] [Google Scholar]
  13. D'Arrigo A., Manera E., Longhi R., Borgese N. The specific subcellular localization of two isoforms of cytochrome b5 suggests novel targeting pathways. J Biol Chem. 1993 Feb 5;268(4):2802–2808. [PubMed] [Google Scholar]
  14. Dietmeier K., Hönlinger A., Bömer U., Dekker P. J., Eckerskorn C., Lottspeich F., Kübrich M., Pfanner N. Tom5 functionally links mitochondrial preprotein receptors to the general import pore. Nature. 1997 Jul 10;388(6638):195–200. doi: 10.1038/40663. [DOI] [PubMed] [Google Scholar]
  15. Drubin D. G., Jones H. D., Wertman K. F. Actin structure and function: roles in mitochondrial organization and morphogenesis in budding yeast and identification of the phalloidin-binding site. Mol Biol Cell. 1993 Dec;4(12):1277–1294. doi: 10.1091/mbc.4.12.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Flinn H. M., Smith D. F. Genomic organisation and expression of a differentially-regulated gene family from Leishmania major. Nucleic Acids Res. 1992 Feb 25;20(4):755–762. doi: 10.1093/nar/20.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Franke E. D., McGreevy P. B., Katz S. P., Sacks D. L. Growth cycle-dependent generation of complement-resistant Leishmania promastigotes. J Immunol. 1985 Apr;134(4):2713–2718. [PubMed] [Google Scholar]
  18. Goldstein L. S., Laymon R. A., McIntosh J. R. A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences. J Cell Biol. 1986 Jun;102(6):2076–2087. doi: 10.1083/jcb.102.6.2076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hauser R., Pypaert M., Häusler T., Horn E. K., Schneider A. In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae. J Cell Sci. 1996 Feb;109(Pt 2):517–523. doi: 10.1242/jcs.109.2.517. [DOI] [PubMed] [Google Scholar]
  20. Heggeness M. H., Simon M., Singer S. J. Association of mitochondria with microtubules in cultured cells. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3863–3866. doi: 10.1073/pnas.75.8.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ivessa A. S., Schneiter R., Kohlwein S. D. Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum. Eur J Cell Biol. 1997 Dec;74(4):399–406. [PubMed] [Google Scholar]
  22. Janiak F., Leber B., Andrews D. W. Assembly of Bcl-2 into microsomal and outer mitochondrial membranes. J Biol Chem. 1994 Apr 1;269(13):9842–9849. [PubMed] [Google Scholar]
  23. Jardim A., Funk V., Caprioli R. M., Olafson R. W. Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein. Biochem J. 1995 Jan 1;305(Pt 1):307–313. doi: 10.1042/bj3050307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kabsch W., Mannherz H. G., Suck D., Pai E. F., Holmes K. C. Atomic structure of the actin:DNase I complex. Nature. 1990 Sep 6;347(6288):37–44. doi: 10.1038/347037a0. [DOI] [PubMed] [Google Scholar]
  25. Kelly J. M., Ward H. M., Miles M. A., Kendall G. A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania. Nucleic Acids Res. 1992 Aug 11;20(15):3963–3969. doi: 10.1093/nar/20.15.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell. 1986 Nov 21;47(4):481–483. doi: 10.1016/0092-8674(86)90609-4. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lewis Carl S. A., Gillete-Ferguson I., Ferguson D. G. An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies. J Histochem Cytochem. 1993 Aug;41(8):1273–1278. doi: 10.1177/41.8.7687266. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Marín M., Muskus C., Ramírez J. R., Arbelaez L. F., Alzate J. F., Berberich C. The gene encoding the metacyclogenesis-associated transcript Mat-1 is conserved in the genus Leishmania and shows a tendency to form dimers upon protein expression. Parasitol Res. 2000 May;86(5):431–435. doi: 10.1007/s004360050690. [DOI] [PubMed] [Google Scholar]
  31. McConville M. J., Turco S. J., Ferguson M. A., Sacks D. L. Developmental modification of lipophosphoglycan during the differentiation of Leishmania major promastigotes to an infectious stage. EMBO J. 1992 Oct;11(10):3593–3600. doi: 10.1002/j.1460-2075.1992.tb05443.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McKean P. G., Delahay R., Pimenta P. F., Smith D. F. Characterisation of a second protein encoded by the differentially regulated LmcDNA16 gene family of Leishmania major. Mol Biochem Parasitol. 1997 Apr;85(2):221–231. doi: 10.1016/s0166-6851(97)02829-6. [DOI] [PubMed] [Google Scholar]
  33. Mottram J. C., Frame M. J., Brooks D. R., Tetley L., Hutchison J. E., Souza A. E., Coombs G. H. The multiple cpb cysteine proteinase genes of Leishmania mexicana encode isoenzymes that differ in their stage regulation and substrate preferences. J Biol Chem. 1997 May 30;272(22):14285–14293. doi: 10.1074/jbc.272.22.14285. [DOI] [PubMed] [Google Scholar]
  34. Nourbakhsh F., Uliana S. R., Smith D. F. Characterisation and expression of a stage-regulated gene of Leishmania major. Mol Biochem Parasitol. 1996 Feb-Mar;76(1-2):201–213. doi: 10.1016/0166-6851(95)02559-6. [DOI] [PubMed] [Google Scholar]
  35. Poupel O., Boleti H., Axisa S., Couture-Tosi E., Tardieux I. Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments. Mol Biol Cell. 2000 Jan;11(1):355–368. doi: 10.1091/mbc.11.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Quijada L., Soto M., Alonso C., Requena J. M. Analysis of post-transcriptional regulation operating on transcription products of the tandemly linked Leishmania infantum hsp70 genes. J Biol Chem. 1997 Feb 14;272(7):4493–4499. doi: 10.1074/jbc.272.7.4493. [DOI] [PubMed] [Google Scholar]
  37. Ramamoorthy R., Donelson J. E., Paetz K. E., Maybodi M., Roberts S. C., Wilson M. E. Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form. J Biol Chem. 1992 Jan 25;267(3):1888–1895. [PubMed] [Google Scholar]
  38. Rizzuto R., Pinton P., Carrington W., Fay F. S., Fogarty K. E., Lifshitz L. M., Tuft R. A., Pozzan T. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science. 1998 Jun 12;280(5370):1763–1766. doi: 10.1126/science.280.5370.1763. [DOI] [PubMed] [Google Scholar]
  39. Sacks D. L., Hieny S., Sher A. Identification of cell surface carbohydrate and antigenic changes between noninfective and infective developmental stages of Leishmania major promastigotes. J Immunol. 1985 Jul;135(1):564–569. [PubMed] [Google Scholar]
  40. Sacks D. L., Perkins P. V. Identification of an infective stage of Leishmania promastigotes. Science. 1984 Mar 30;223(4643):1417–1419. doi: 10.1126/science.6701528. [DOI] [PubMed] [Google Scholar]
  41. Safer D., Elzinga M., Nachmias V. T. Thymosin beta 4 and Fx, an actin-sequestering peptide, are indistinguishable. J Biol Chem. 1991 Mar 5;266(7):4029–4032. [PubMed] [Google Scholar]
  42. Schneider A., Martin J., Agabian N. A nuclear encoded tRNA of Trypanosoma brucei is imported into mitochondria. Mol Cell Biol. 1994 Apr;14(4):2317–2322. doi: 10.1128/mcb.14.4.2317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Schneider H. C., Berthold J., Bauer M. F., Dietmeier K., Guiard B., Brunner M., Neupert W. Mitochondrial Hsp70/MIM44 complex facilitates protein import. Nature. 1994 Oct 27;371(6500):768–774. doi: 10.1038/371768a0. [DOI] [PubMed] [Google Scholar]
  44. Schneider P., Rosat J. P., Bouvier J., Louis J., Bordier C. Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages. Exp Parasitol. 1992 Sep;75(2):196–206. doi: 10.1016/0014-4894(92)90179-e. [DOI] [PubMed] [Google Scholar]
  45. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  46. Searle S., McCrossan M. V., Smith D. F. Expression of a mitochondrial stress protein in the protozoan parasite Leishmania major. J Cell Sci. 1993 Apr;104(Pt 4):1091–1100. doi: 10.1242/jcs.104.4.1091. [DOI] [PubMed] [Google Scholar]
  47. Soto M., Quijada L., Alonso C., Requena J. M. Histone synthesis in Leishmania infantum is tightly linked to DNA replication by a translational control. Biochem J. 2000 Feb 15;346(Pt 1):99–105. [PMC free article] [PubMed] [Google Scholar]
  48. Tokuyasu K. T. Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy. Histochem J. 1989 Mar;21(3):163–171. doi: 10.1007/BF01007491. [DOI] [PubMed] [Google Scholar]
  49. Uliana S. R., Goyal N., Freymüller E., Smith D. F. Leishmania: overexpression and comparative structural analysis of the stage-regulated meta 1 gene. Exp Parasitol. 1999 Jul;92(3):183–191. doi: 10.1006/expr.1999.4410. [DOI] [PubMed] [Google Scholar]
  50. Wilkinson B. M., Esnault Y., Craven R. A., Skiba F., Fieschi J., K'epès F., Stirling C. J. Molecular architecture of the ER translocase probed by chemical crosslinking of Sss1p to complementary fragments of Sec61p. EMBO J. 1997 Aug 1;16(15):4549–4559. doi: 10.1093/emboj/16.15.4549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. de Gunzburg J., Riehl R., Weinberg R. A. Identification of a protein associated with p21ras by chemical crosslinking. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4007–4011. doi: 10.1073/pnas.86.11.4007. [DOI] [PMC free article] [PubMed] [Google Scholar]

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