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. 1996 Oct;7(10):1623–1638. doi: 10.1091/mbc.7.10.1623

A role for a Rab4-like GTPase in endocytosis and in regulation of contractile vacuole structure and function in Dictyostelium discoideum.

J Bush 1, L Temesvari 1, J Rodriguez-Paris 1, G Buczynski 1, J Cardelli 1
PMCID: PMC276010  PMID: 8898366

Abstract

The small Mr Rab4-like GTPase, RabD, localizes to the endosomal pathway and the contractile vacuole membrane system in Dictyostelium discoideum. Stably transformed cell lines overexpressing a dominant negative functioning RabD internalized fluid phase marker at 50% of the rate of wild-type cells. Mutant cells were also slower at recycling internalized fluid. Microscopic and biochemical approaches indicated that the transport of fluid to large postlysosome vacuoles was delayed in mutant cells, resulting in an accumulation in acidic smaller vesicles, probably lysosomes. Also, RabD N121I-expressing cell lines missorted a small but significant percentage of newly synthesized lysosomal alpha-mannosidase precursor polypeptides. However, the majority of the newly synthesized alpha-mannosidase was transported with normal kinetics and correctly delivered to lysosomes. Subcellular fractionation and immunofluorescent microscopy indicated that in mutant cells contractile vacuole membrane proteins were associated with compartments morphologically distinct from the normal reticular network. Osmotic tests revealed that the contractile vacuole functioned inefficiently in mutant cells. Our results suggest that RabD regulates membrane traffic along the endosomal pathway, and that this GTPase may play a role in regulating the structure and function of the contractile vacuole system by facilitating communication with the endosomal pathway.

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Selected References

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

  1. Aley S. B., Cohn Z. A., Scott W. A. Endocytosis in Entamoeba histolytica. Evidence for a unique non-acidified compartment. J Exp Med. 1984 Sep 1;160(3):724–737. doi: 10.1084/jem.160.3.724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aubry L., Klein G., Martiel J. L., Satre M. Kinetics of endosomal pH evolution in Dictyostelium discoideum amoebae. Study by fluorescence spectroscopy. J Cell Sci. 1993 Jul;105(Pt 3):861–866. doi: 10.1242/jcs.105.3.861. [DOI] [PubMed] [Google Scholar]
  3. Bucci C., Parton R. G., Mather I. H., Stunnenberg H., Simons K., Hoflack B., Zerial M. The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway. Cell. 1992 Sep 4;70(5):715–728. doi: 10.1016/0092-8674(92)90306-w. [DOI] [PubMed] [Google Scholar]
  4. Bush J. M., Cardelli J. A. Processing, transport, and secretion of the lysosomal enzyme acid phosphatase in Dictyostelium discoideum. J Biol Chem. 1989 May 5;264(13):7630–7636. [PubMed] [Google Scholar]
  5. Bush J., Franek K., Daniel J., Spiegelman G. B., Weeks G., Cardelli J. Cloning and characterization of five novel Dictyostelium discoideum rab-related genes. Gene. 1993 Dec 22;136(1-2):55–60. doi: 10.1016/0378-1119(93)90447-b. [DOI] [PubMed] [Google Scholar]
  6. Bush J., Nolta K., Rodriguez-Paris J., Kaufmann N., O'Halloran T., Ruscetti T., Temesvari L., Steck T., Cardelli J. A Rab4-like GTPase in Dictyostelium discoideum colocalizes with V-H(+)-ATPases in reticular membranes of the contractile vacuole complex and in lysosomes. J Cell Sci. 1994 Oct;107(Pt 10):2801–2812. doi: 10.1242/jcs.107.10.2801. [DOI] [PubMed] [Google Scholar]
  7. Cardelli J. A., Bush J. M., Ebert D., Freeze H. H. Sulfated N-linked oligosaccharides affect secretion but are not essential for the transport, proteolytic processing, and sorting of lysosomal enzymes in Dictyostelium discoideum. J Biol Chem. 1990 May 25;265(15):8847–8853. [PubMed] [Google Scholar]
  8. Cardelli J. A., Golumbeski G. S., Dimond R. L. Lysosomal enzymes in Dictyostelium discoideum are transported to lysosomes at distinctly different rates. J Cell Biol. 1986 Apr;102(4):1264–1270. doi: 10.1083/jcb.102.4.1264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cardelli J. A., Golumbeski G. S., Woychik N. A., Ebert D. L., Mierendorf R. C., Dimond R. L. Defining the intracellular localization pathways followed by lysosomal enzymes in Dictyostelium discoideum. Methods Cell Biol. 1987;28:139–155. doi: 10.1016/s0091-679x(08)61641-6. [DOI] [PubMed] [Google Scholar]
  10. Cardelli J. A., Richardson J., Miears D. Role of acidic intracellular compartments in the biosynthesis of Dictyostelium lysosomal enzymes. The weak bases ammonium chloride and chloroquine differentially affect proteolytic processing and sorting. J Biol Chem. 1989 Feb 25;264(6):3454–3463. [PubMed] [Google Scholar]
  11. Cardelli J. A., Schatzle J., Bush J. M., Richardson J., Ebert D., Freeze H. Biochemical and genetic analysis of the biosynthesis, sorting, and secretion of Dictyostelium lysosomal enzymes. Dev Genet. 1990;11(5-6):454–462. doi: 10.1002/dvg.1020110522. [DOI] [PubMed] [Google Scholar]
  12. Chavrier P., Parton R. G., Hauri H. P., Simons K., Zerial M. Localization of low molecular weight GTP binding proteins to exocytic and endocytic compartments. Cell. 1990 Jul 27;62(2):317–329. doi: 10.1016/0092-8674(90)90369-p. [DOI] [PubMed] [Google Scholar]
  13. Chen Y. T., Holcomb C., Moore H. P. Expression and localization of two low molecular weight GTP-binding proteins, Rab8 and Rab10, by epitope tag. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6508–6512. doi: 10.1073/pnas.90.14.6508. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Feng Y., Press B., Wandinger-Ness A. Rab 7: an important regulator of late endocytic membrane traffic. J Cell Biol. 1995 Dec;131(6 Pt 1):1435–1452. doi: 10.1083/jcb.131.6.1435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fok A. K., Clarke M., Ma L., Allen R. D. Vacuolar H(+)-ATPase of Dictyostelium discoideum. A monoclonal antibody study. J Cell Sci. 1993 Dec;106(Pt 4):1103–1113. doi: 10.1242/jcs.106.4.1103. [DOI] [PubMed] [Google Scholar]
  16. Freeze H. H., Bush J. M., Cardelli J. Biochemical and genetic analysis of an antigenic determinant found on N-linked oligosaccharides in Dictyostelium. Dev Genet. 1990;11(5-6):463–472. doi: 10.1002/dvg.1020110523. [DOI] [PubMed] [Google Scholar]
  17. Freeze H. H. Modifications of lysosomal enzymes in Dictyostelium discoideum. Mol Cell Biochem. 1986 Nov-Dec;72(1-2):47–65. doi: 10.1007/BF00230635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Heuser J., Zhu Q., Clarke M. Proton pumps populate the contractile vacuoles of Dictyostelium amoebae. J Cell Biol. 1993 Jun;121(6):1311–1327. doi: 10.1083/jcb.121.6.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Li G., D'Souza-Schorey C., Barbieri M. A., Roberts R. L., Klippel A., Williams L. T., Stahl P. D. Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5. Proc Natl Acad Sci U S A. 1995 Oct 24;92(22):10207–10211. doi: 10.1073/pnas.92.22.10207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lombardi D., Soldati T., Riederer M. A., Goda Y., Zerial M., Pfeffer S. R. Rab9 functions in transport between late endosomes and the trans Golgi network. EMBO J. 1993 Feb;12(2):677–682. doi: 10.1002/j.1460-2075.1993.tb05701.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Luna E. J., Condeelis J. S. Actin-associated proteins in Dictyostelium discoideum. Dev Genet. 1990;11(5-6):328–332. doi: 10.1002/dvg.1020110503. [DOI] [PubMed] [Google Scholar]
  23. Mierendorf R. C., Jr, Cardelli J. A., Dimond R. L. Pathways involved in targeting and secretion of a lysosomal enzyme in Dictyostelium discoideum. J Cell Biol. 1985 May;100(5):1777–1787. doi: 10.1083/jcb.100.5.1777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moniakis J., Coukell M. B., Forer A. Molecular cloning of an intracellular P-type ATPase from Dictyostelium that is up-regulated in calcium-adapted cells. J Biol Chem. 1995 Nov 24;270(47):28276–28281. doi: 10.1074/jbc.270.47.28276. [DOI] [PubMed] [Google Scholar]
  25. Méresse S., Gorvel J. P., Chavrier P. The rab7 GTPase resides on a vesicular compartment connected to lysosomes. J Cell Sci. 1995 Nov;108(Pt 11):3349–3358. doi: 10.1242/jcs.108.11.3349. [DOI] [PubMed] [Google Scholar]
  26. Nolta K. V., Padh H., Steck T. L. Acidosomes from Dictyostelium. Initial biochemical characterization. J Biol Chem. 1991 Sep 25;266(27):18318–18323. [PubMed] [Google Scholar]
  27. Nolta K. V., Rodriguez-Paris J. M., Steck T. L. Analysis of successive endocytic compartments isolated from Dictyostelium discoideum by magnetic fractionation. Biochim Biophys Acta. 1994 Nov 10;1224(2):237–246. doi: 10.1016/0167-4889(94)90196-1. [DOI] [PubMed] [Google Scholar]
  28. Nolta K. V., Steck T. L. Isolation and initial characterization of the bipartite contractile vacuole complex from Dictyostelium discoideum. J Biol Chem. 1994 Jan 21;269(3):2225–2233. [PubMed] [Google Scholar]
  29. Novak K. D., Peterson M. D., Reedy M. C., Titus M. A. Dictyostelium myosin I double mutants exhibit conditional defects in pinocytosis. J Cell Biol. 1995 Dec;131(5):1205–1221. doi: 10.1083/jcb.131.5.1205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. O'Halloran T. J., Anderson R. G. Clathrin heavy chain is required for pinocytosis, the presence of large vacuoles, and development in Dictyostelium. J Cell Biol. 1992 Sep;118(6):1371–1377. doi: 10.1083/jcb.118.6.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Padh H., Ha J., Lavasa M., Steck T. L. A post-lysosomal compartment in Dictyostelium discoideum. J Biol Chem. 1993 Mar 25;268(9):6742–6747. [PubMed] [Google Scholar]
  32. Padh H., Lavasa M., Steck T. L. Endosomes are acidified by association with discrete proton-pumping vacuoles in Dictyostelium. J Biol Chem. 1991 Mar 25;266(9):5514–5520. [PubMed] [Google Scholar]
  33. Padh H., Lavasa M., Steck T. L. Reconstitution of the association of endocytic vacuoles and acidosomes from Dictyostelium. J Biol Chem. 1991 Jul 5;266(19):12123–12126. [PubMed] [Google Scholar]
  34. Padh H., Tanjore S. Localization of cyclic-AMP receptors with acidosomes in Dictyostelium discoideum. FEBS Lett. 1995 Jul 17;368(2):358–362. doi: 10.1016/0014-5793(95)00656-t. [DOI] [PubMed] [Google Scholar]
  35. Pfeffer S. R. Rab GTPases: master regulators of membrane trafficking. Curr Opin Cell Biol. 1994 Aug;6(4):522–526. doi: 10.1016/0955-0674(94)90071-x. [DOI] [PubMed] [Google Scholar]
  36. Richardson J. M., Woychik N. A., Ebert D. L., Dimond R. L., Cardelli J. A. Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum. J Cell Biol. 1988 Dec;107(6 Pt 1):2097–2107. doi: 10.1083/jcb.107.6.2097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rodriguez-Paris J. M., Nolta K. V., Steck T. L. Characterization of lysosomes isolated from Dictyostelium discoideum by magnetic fractionation. J Biol Chem. 1993 Apr 25;268(12):9110–9116. [PubMed] [Google Scholar]
  38. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  39. Ruscetti T., Cardelli J. A., Niswonger M. L., O'Halloran T. J. Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum. J Cell Biol. 1994 Jul;126(2):343–352. doi: 10.1083/jcb.126.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Salminen A., Novick P. J. A ras-like protein is required for a post-Golgi event in yeast secretion. Cell. 1987 May 22;49(4):527–538. doi: 10.1016/0092-8674(87)90455-7. [DOI] [PubMed] [Google Scholar]
  41. Saxe S. A., Kimmel A. R. SAS1 and SAS2, GTP-binding protein genes in Dictyostelium discoideum with sequence similarities to essential genes in Saccharomyces cerevisiae. Mol Cell Biol. 1990 May;10(5):2367–2378. doi: 10.1128/mcb.10.5.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schimmöller F., Riezman H. Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast. J Cell Sci. 1993 Nov;106(Pt 3):823–830. doi: 10.1242/jcs.106.3.823. [DOI] [PubMed] [Google Scholar]
  43. Temesvari L. A., Bush J. M., Peterson M. D., Novak K. D., Titus M. A., Cardelli J. A. Examination of the endosomal and lysosomal pathways in Dictyostelium discoideum myosin I mutants. J Cell Sci. 1996 Mar;109(Pt 3):663–673. doi: 10.1242/jcs.109.3.663. [DOI] [PubMed] [Google Scholar]
  44. Temesvari L. A., Rodriguez-Paris J. M., Bush J. M., Zhang L., Cardelli J. A. Involvement of the vacuolar proton-translocating ATPase in multiple steps of the endo-lysosomal system and in the contractile vacuole system of Dictyostelium discoideum. J Cell Sci. 1996 Jun;109(Pt 6):1479–1495. doi: 10.1242/jcs.109.6.1479. [DOI] [PubMed] [Google Scholar]
  45. Temesvari L., Rodriguez-Paris J., Bush J., Steck T. L., Cardelli J. Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits. J Biol Chem. 1994 Oct 14;269(41):25719–25727. [PubMed] [Google Scholar]
  46. Tisdale E. J., Bourne J. R., Khosravi-Far R., Der C. J., Balch W. E. GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex. J Cell Biol. 1992 Nov;119(4):749–761. doi: 10.1083/jcb.119.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Van Der Sluijs P., Hull M., Zahraoui A., Tavitian A., Goud B., Mellman I. The small GTP-binding protein rab4 is associated with early endosomes. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6313–6317. doi: 10.1073/pnas.88.14.6313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Walworth N. C., Goud B., Kabcenell A. K., Novick P. J. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. doi: 10.1002/j.1460-2075.1989.tb03560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wichmann H., Hengst L., Gallwitz D. Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p). Cell. 1992 Dec 24;71(7):1131–1142. doi: 10.1016/s0092-8674(05)80062-5. [DOI] [PubMed] [Google Scholar]
  50. Wood L., Kaplan A. Transit of alpha-mannosidase during its maturation in Dictyostelium discoideum. J Cell Biol. 1985 Dec;101(6):2063–2069. doi: 10.1083/jcb.101.6.2063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhu Q., Liu T., Clarke M. Calmodulin and the contractile vacuole complex in mitotic cells of Dictyostelium discoideum. J Cell Sci. 1993 Apr;104(Pt 4):1119–1127. doi: 10.1242/jcs.104.4.1119. [DOI] [PubMed] [Google Scholar]
  52. van der Sluijs P., Hull M., Webster P., Mâle P., Goud B., Mellman I. The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell. 1992 Sep 4;70(5):729–740. doi: 10.1016/0092-8674(92)90307-x. [DOI] [PubMed] [Google Scholar]

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