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. 1997 Jul;8(7):1361–1375. doi: 10.1091/mbc.8.7.1361

The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

V Moreau 1, J M Galan 1, G Devilliers 1, R Haguenauer-Tsapis 1, B Winsor 1
PMCID: PMC276158  PMID: 9243513

Abstract

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

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Selected References

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

  1. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bénédetti H., Raths S., Crausaz F., Riezman H. The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast. Mol Biol Cell. 1994 Sep;5(9):1023–1037. doi: 10.1091/mbc.5.9.1023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  4. Cleves A. E., Novick P. J., Bankaitis V. A. Mutations in the SAC1 gene suppress defects in yeast Golgi and yeast actin function. J Cell Biol. 1989 Dec;109(6 Pt 1):2939–2950. doi: 10.1083/jcb.109.6.2939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Davis N. G., Horecka J. L., Sprague G. F., Jr Cis- and trans-acting functions required for endocytosis of the yeast pheromone receptors. J Cell Biol. 1993 Jul;122(1):53–65. doi: 10.1083/jcb.122.1.53. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donnelly S. F., Pocklington M. J., Pallotta D., Orr E. A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae. Mol Microbiol. 1993 Nov;10(3):585–596. doi: 10.1111/j.1365-2958.1993.tb00930.x. [DOI] [PubMed] [Google Scholar]
  7. Drubin D. G. Development of cell polarity in budding yeast. Cell. 1991 Jun 28;65(7):1093–1096. doi: 10.1016/0092-8674(91)90001-f. [DOI] [PubMed] [Google Scholar]
  8. Ecker D. J., Khan M. I., Marsh J., Butt T. R., Crooke S. T. Chemical synthesis and expression of a cassette adapted ubiquitin gene. J Biol Chem. 1987 Mar 15;262(8):3524–3527. [PubMed] [Google Scholar]
  9. Egner R., Kuchler K. The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole. FEBS Lett. 1996 Jan 8;378(2):177–181. doi: 10.1016/0014-5793(95)01450-0. [DOI] [PubMed] [Google Scholar]
  10. Field C., Schekman R. Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J Cell Biol. 1980 Jul;86(1):123–128. doi: 10.1083/jcb.86.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Galan J. M., Moreau V., Andre B., Volland C., Haguenauer-Tsapis R. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem. 1996 May 3;271(18):10946–10952. doi: 10.1074/jbc.271.18.10946. [DOI] [PubMed] [Google Scholar]
  12. Geli M. I., Riezman H. Role of type I myosins in receptor-mediated endocytosis in yeast. Science. 1996 Apr 26;272(5261):533–535. doi: 10.1126/science.272.5261.533. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Goldstein A., Lampen J. O. Beta-D-fructofuranoside fructohydrolase from yeast. Methods Enzymol. 1975;42:504–511. doi: 10.1016/0076-6879(75)42159-0. [DOI] [PubMed] [Google Scholar]
  15. Goodson H. V., Anderson B. L., Warrick H. M., Pon L. A., Spudich J. A. Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): myosin I proteins are required for polarization of the actin cytoskeleton. J Cell Biol. 1996 Jun;133(6):1277–1291. doi: 10.1083/jcb.133.6.1277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Govindan B., Bowser R., Novick P. The role of Myo2, a yeast class V myosin, in vesicular transport. J Cell Biol. 1995 Mar;128(6):1055–1068. doi: 10.1083/jcb.128.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Graham T. R., Emr S. D. Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol. 1991 Jul;114(2):207–218. doi: 10.1083/jcb.114.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Harsay E., Bretscher A. Parallel secretory pathways to the cell surface in yeast. J Cell Biol. 1995 Oct;131(2):297–310. doi: 10.1083/jcb.131.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hein C., Springael J. Y., Volland C., Haguenauer-Tsapis R., André B. NPl1, an essential yeast gene involved in induced degradation of Gap1 and Fur4 permeases, encodes the Rsp5 ubiquitin-protein ligase. Mol Microbiol. 1995 Oct;18(1):77–87. doi: 10.1111/j.1365-2958.1995.mmi_18010077.x. [DOI] [PubMed] [Google Scholar]
  20. Hicke L., Riezman H. Ubiquitination of a yeast plasma membrane receptor signals its ligand-stimulated endocytosis. Cell. 1996 Jan 26;84(2):277–287. doi: 10.1016/s0092-8674(00)80982-4. [DOI] [PubMed] [Google Scholar]
  21. Hochstrasser M., Ellison M. J., Chau V., Varshavsky A. The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4606–4610. doi: 10.1073/pnas.88.11.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Holtzman D. A., Wertman K. F., Drubin D. G. Mapping actin surfaces required for functional interactions in vivo. J Cell Biol. 1994 Jul;126(2):423–432. doi: 10.1083/jcb.126.2.423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Holtzman D. A., Yang S., Drubin D. G. Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae. J Cell Biol. 1993 Aug;122(3):635–644. doi: 10.1083/jcb.122.3.635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jansen R. P., Dowzer C., Michaelis C., Galova M., Nasmyth K. Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins. Cell. 1996 Mar 8;84(5):687–697. doi: 10.1016/s0092-8674(00)81047-8. [DOI] [PubMed] [Google Scholar]
  25. Johnston G. C., Prendergast J. A., Singer R. A. The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol. 1991 May;113(3):539–551. doi: 10.1083/jcb.113.3.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Julius D., Schekman R., Thorner J. Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell. 1984 Feb;36(2):309–318. doi: 10.1016/0092-8674(84)90224-1. [DOI] [PubMed] [Google Scholar]
  27. Jund R., Weber E., Chevallier M. R. Primary structure of the uracil transport protein of Saccharomyces cerevisiae. Eur J Biochem. 1988 Jan 15;171(1-2):417–424. doi: 10.1111/j.1432-1033.1988.tb13806.x. [DOI] [PubMed] [Google Scholar]
  28. Kelleher J. F., Atkinson S. J., Pollard T. D. Sequences, structural models, and cellular localization of the actin-related proteins Arp2 and Arp3 from Acanthamoeba. J Cell Biol. 1995 Oct;131(2):385–397. doi: 10.1083/jcb.131.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kölling R., Hollenberg C. P. The ABC-transporter Ste6 accumulates in the plasma membrane in a ubiquitinated form in endocytosis mutants. EMBO J. 1994 Jul 15;13(14):3261–3271. doi: 10.1002/j.1460-2075.1994.tb06627.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kübler E., Riezman H. Actin and fimbrin are required for the internalization step of endocytosis in yeast. EMBO J. 1993 Jul;12(7):2855–2862. doi: 10.1002/j.1460-2075.1993.tb05947.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kübler E., Schimmöller F., Riezman H. Calcium-independent calmodulin requirement for endocytosis in yeast. EMBO J. 1994 Dec 1;13(23):5539–5546. doi: 10.1002/j.1460-2075.1994.tb06891.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lai K., Bolognese C. P., Swift S., McGraw P. Regulation of inositol transport in Saccharomyces cerevisiae involves inositol-induced changes in permease stability and endocytic degradation in the vacuole. J Biol Chem. 1995 Feb 10;270(6):2525–2534. doi: 10.1074/jbc.270.6.2525. [DOI] [PubMed] [Google Scholar]
  33. Liu H., Bretscher A. Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport. J Cell Biol. 1992 Jul;118(2):285–299. doi: 10.1083/jcb.118.2.285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Machesky L. M., Atkinson S. J., Ampe C., Vandekerckhove J., Pollard T. D. Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose. J Cell Biol. 1994 Oct;127(1):107–115. doi: 10.1083/jcb.127.1.107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moreau V., Madania A., Martin R. P., Winson B. The Saccharomyces cerevisiae actin-related protein Arp2 is involved in the actin cytoskeleton. J Cell Biol. 1996 Jul;134(1):117–132. doi: 10.1083/jcb.134.1.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mulholland J., Preuss D., Moon A., Wong A., Drubin D., Botstein D. Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J Cell Biol. 1994 Apr;125(2):381–391. doi: 10.1083/jcb.125.2.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Munn A. L., Stevenson B. J., Geli M. I., Riezman H. end5, end6, and end7: mutations that cause actin delocalization and block the internalization step of endocytosis in Saccharomyces cerevisiae. Mol Biol Cell. 1995 Dec;6(12):1721–1742. doi: 10.1091/mbc.6.12.1721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nelson K. K., Holmer M., Lemmon S. K. SCD5, a suppressor of clathrin deficiency, encodes a novel protein with a late secretory function in yeast. Mol Biol Cell. 1996 Feb;7(2):245–260. doi: 10.1091/mbc.7.2.245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  40. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  41. Novick P., Osmond B. C., Botstein D. Suppressors of yeast actin mutations. Genetics. 1989 Apr;121(4):659–674. doi: 10.1093/genetics/121.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Novick P., Schekman R. Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1858–1862. doi: 10.1073/pnas.76.4.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Raths S., Rohrer J., Crausaz F., Riezman H. end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae. J Cell Biol. 1993 Jan;120(1):55–65. doi: 10.1083/jcb.120.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Riballo E., Herweijer M., Wolf D. H., Lagunas R. Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis. J Bacteriol. 1995 Oct;177(19):5622–5627. doi: 10.1128/jb.177.19.5622-5627.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Riezman H. Yeast endocytosis. Trends Cell Biol. 1993 Aug;3(8):273–277. doi: 10.1016/0962-8924(93)90056-7. [DOI] [PubMed] [Google Scholar]
  46. Roth A. F., Davis N. G. Ubiquitination of the yeast a-factor receptor. J Cell Biol. 1996 Aug;134(3):661–674. doi: 10.1083/jcb.134.3.661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sachs A. B., Deardorff J. A. Translation initiation requires the PAB-dependent poly(A) ribonuclease in yeast. Cell. 1992 Sep 18;70(6):961–973. doi: 10.1016/0092-8674(92)90246-9. [DOI] [PubMed] [Google Scholar]
  48. Schwob E., Martin R. P. New yeast actin-like gene required late in the cell cycle. Nature. 1992 Jan 9;355(6356):179–182. doi: 10.1038/355179a0. [DOI] [PubMed] [Google Scholar]
  49. Singer B., Riezman H. Detection of an intermediate compartment involved in transport of alpha-factor from the plasma membrane to the vacuole in yeast. J Cell Biol. 1990 Jun;110(6):1911–1922. doi: 10.1083/jcb.110.6.1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sivadon P., Bauer F., Aigle M., Crouzet M. Actin cytoskeleton and budding pattern are altered in the yeast rvs161 mutant: the Rvs161 protein shares common domains with the brain protein amphiphysin. Mol Gen Genet. 1995 Feb 20;246(4):485–495. doi: 10.1007/BF00290452. [DOI] [PubMed] [Google Scholar]
  51. Tang H. Y., Cai M. The EH-domain-containing protein Pan1 is required for normal organization of the actin cytoskeleton in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Sep;16(9):4897–4914. doi: 10.1128/mcb.16.9.4897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tschopp J., Esmon P. C., Schekman R. Defective plasma membrane assembly in yeast secretory mutants. J Bacteriol. 1984 Dec;160(3):966–970. doi: 10.1128/jb.160.3.966-970.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Vida T. A., Huyer G., Emr S. D. Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment. J Cell Biol. 1993 Jun;121(6):1245–1256. doi: 10.1083/jcb.121.6.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Volland C., Garnier C., Haguenauer-Tsapis R. In vivo phosphorylation of the yeast uracil permease. J Biol Chem. 1992 Nov 25;267(33):23767–23771. [PubMed] [Google Scholar]
  56. Volland C., Urban-Grimal D., Géraud G., Haguenauer-Tsapis R. Endocytosis and degradation of the yeast uracil permease under adverse conditions. J Biol Chem. 1994 Apr 1;269(13):9833–9841. [PubMed] [Google Scholar]
  57. Welch M. D., Holtzman D. A., Drubin D. G. The yeast actin cytoskeleton. Curr Opin Cell Biol. 1994 Feb;6(1):110–119. doi: 10.1016/0955-0674(94)90124-4. [DOI] [PubMed] [Google Scholar]
  58. Wendland B., McCaffery J. M., Xiao Q., Emr S. D. A novel fluorescence-activated cell sorter-based screen for yeast endocytosis mutants identifies a yeast homologue of mammalian eps15. J Cell Biol. 1996 Dec;135(6 Pt 1):1485–1500. doi: 10.1083/jcb.135.6.1485. [DOI] [PMC free article] [PubMed] [Google Scholar]

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