Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1997 Jul;8(7):1317–1327. doi: 10.1091/mbc.8.7.1317

The ankyrin repeat-containing protein Akr1p is required for the endocytosis of yeast pheromone receptors.

S A Givan 1, G F Sprague Jr 1
PMCID: PMC276155  PMID: 9243510

Abstract

The Saccharomyces cerevisiae a-factor receptor (Ste3p) requires its C-terminal cytoplasmic tail for endocytosis. Wild-type receptor is delivered to the cell surface via the secretory pathway but remains there only briefly before being internalized and delivered to the vacuole for degradation. Receptors lacking all or part of the cytoplasmic tail are not subject to this constitutive endocytosis. We used the cytoplasmic tail of Ste3p as bait in the two-hybrid system in an effort to identify other proteins involved in endocytosis. One protein identified was Akr1p, an ankyrin repeat-containing protein. We applied three criteria to demonstrate that Akr1p is involved in the constitutive endocytosis of Ste3p. First, when receptor synthesis is shut off, akr1 delta cells retain the ability to mate longer than do AKR1 cells. Second, Ste3p half-life is increased by greater than 5-fold in akr1 delta cells compared with AKR1 cells. Third, after a pulse of synthesis, newly synthesized receptor remains at the cell surface in akr1 delta mutants, whereas it is rapidly internalized in AKR1 cells. Specifically, in akr1 delta mutants, newly synthesized receptor is accessible to exogenous protease, and by indirect immunofluorescence, the receptor is located at the cell surface. akr1 delta cells are also defective for endocytosis of the alpha-factor receptor (Ste2p). Despite the block to constitutive endocytosis exhibited by akr1 delta cells, they are competent to carry out ligand-mediated endocytosis of Ste3p. In contrast, akr1 delta cells cannot carry out ligand-mediated endocytosis of Ste2p. We discuss the implications for Akr1p function in endocytosis and suggest a link to the regulation of ADP-ribosylation proteins (Arf proteins).

Full text

PDF
1317

Images in this article

1320Image
on p.1320
1322Image
on p.1322
1322Image
on p.1322
1323Image
on p.1323
1324Image
on p.1324
1324Image
on p.1324
1325Image
on p.1325

Selected References

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

  1. 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]
  2. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  3. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Colombo M. I., Inglese J., D'Souza-Schorey C., Beron W., Stahl P. D. Heterotrimeric G proteins interact with the small GTPase ARF. Possibilities for the regulation of vesicular traffic. J Biol Chem. 1995 Oct 13;270(41):24564–24571. doi: 10.1074/jbc.270.41.24564. [DOI] [PubMed] [Google Scholar]
  5. D'Souza-Schorey C., Li G., Colombo M. I., Stahl P. D. A regulatory role for ARF6 in receptor-mediated endocytosis. Science. 1995 Feb 24;267(5201):1175–1178. doi: 10.1126/science.7855600. [DOI] [PubMed] [Google Scholar]
  6. D'Souza-Schorey C., Stahl P. D. Myristoylation is required for the intracellular localization and endocytic function of ARF6. Exp Cell Res. 1995 Nov;221(1):153–159. doi: 10.1006/excr.1995.1362. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Gammie A. E., Kurihara L. J., Vallee R. B., Rose M. D. DNM1, a dynamin-related gene, participates in endosomal trafficking in yeast. J Cell Biol. 1995 Aug;130(3):553–566. doi: 10.1083/jcb.130.3.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. 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]
  12. Jenness D. D., Spatrick P. Down regulation of the alpha-factor pheromone receptor in S. cerevisiae. Cell. 1986 Aug 1;46(3):345–353. doi: 10.1016/0092-8674(86)90655-0. [DOI] [PubMed] [Google Scholar]
  13. Kao L. R., Peterson J., Ji R., Bender L., Bender A. Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jan;16(1):168–178. doi: 10.1128/mcb.16.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Konopka J. B., Jenness D. D., Hartwell L. H. The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone. Cell. 1988 Aug 26;54(5):609–620. doi: 10.1016/s0092-8674(88)80005-9. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Lee F. J., Stevens L. A., Kao Y. L., Moss J., Vaughan M. Characterization of a glucose-repressible ADP-ribosylation factor 3 (ARF3) from Saccharomyces cerevisiae. J Biol Chem. 1994 Aug 19;269(33):20931–20937. [PubMed] [Google Scholar]
  18. 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]
  19. Moss J., Vaughan M. Structure and function of ARF proteins: activators of cholera toxin and critical components of intracellular vesicular transport processes. J Biol Chem. 1995 May 26;270(21):12327–12330. doi: 10.1074/jbc.270.21.12327. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Payne G. S., Baker D., van Tuinen E., Schekman R. Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast. J Cell Biol. 1988 May;106(5):1453–1461. doi: 10.1083/jcb.106.5.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peters P. J., Hsu V. W., Ooi C. E., Finazzi D., Teal S. B., Oorschot V., Donaldson J. G., Klausner R. D. Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J Cell Biol. 1995 Mar;128(6):1003–1017. doi: 10.1083/jcb.128.6.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Piper R. C., Cooper A. A., Yang H., Stevens T. H. VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J Cell Biol. 1995 Nov;131(3):603–617. doi: 10.1083/jcb.131.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Poon P. P., Wang X., Rotman M., Huber I., Cukierman E., Cassel D., Singer R. A., Johnston G. C. Saccharomyces cerevisiae Gcs1 is an ADP-ribosylation factor GTPase-activating protein. Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10074–10077. doi: 10.1073/pnas.93.19.10074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pryciak P. M., Hartwell L. H. AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction. Mol Cell Biol. 1996 Jun;16(6):2614–2626. doi: 10.1128/mcb.16.6.2614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Reneke J. E., Blumer K. J., Courchesne W. E., Thorner J. The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain. Cell. 1988 Oct 21;55(2):221–234. doi: 10.1016/0092-8674(88)90045-1. [DOI] [PubMed] [Google Scholar]
  28. Rohrer J., Bénédetti H., Zanolari B., Riezman H. Identification of a novel sequence mediating regulated endocytosis of the G protein-coupled alpha-pheromone receptor in yeast. Mol Biol Cell. 1993 May;4(5):511–521. doi: 10.1091/mbc.4.5.511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  31. Schandel K. A., Jenness D. D. Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor. Mol Cell Biol. 1994 Nov;14(11):7245–7255. doi: 10.1128/mcb.14.11.7245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Singer-Krüger B., Stenmark H., Düsterhöft A., Philippsen P., Yoo J. S., Gallwitz D., Zerial M. Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J Cell Biol. 1994 Apr;125(2):283–298. doi: 10.1083/jcb.125.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Singer-Krüger B., Stenmark H., Zerial M. Yeast Ypt51p and mammalian Rab5: counterparts with similar function in the early endocytic pathway. J Cell Sci. 1995 Nov;108(Pt 11):3509–3521. doi: 10.1242/jcs.108.11.3509. [DOI] [PubMed] [Google Scholar]
  35. Sprague G. F., Jr Assay of yeast mating reaction. Methods Enzymol. 1991;194:77–93. doi: 10.1016/0076-6879(91)94008-z. [DOI] [PubMed] [Google Scholar]
  36. Stearns T., Kahn R. A., Botstein D., Hoyt M. A. ADP ribosylation factor is an essential protein in Saccharomyces cerevisiae and is encoded by two genes. Mol Cell Biol. 1990 Dec;10(12):6690–6699. doi: 10.1128/mcb.10.12.6690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stearns T., Willingham M. C., Botstein D., Kahn R. A. ADP-ribosylation factor is functionally and physically associated with the Golgi complex. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1238–1242. doi: 10.1073/pnas.87.3.1238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tan P. K., Davis N. G., Sprague G. F., Payne G. S. Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast. J Cell Biol. 1993 Dec;123(6 Pt 2):1707–1716. doi: 10.1083/jcb.123.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tan P. K., Howard J. P., Payne G. S. The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae. J Cell Biol. 1996 Dec;135(6 Pt 2):1789–1800. doi: 10.1083/jcb.135.6.1789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Wang X., Hoekstra M. F., DeMaggio A. J., Dhillon N., Vancura A., Kuret J., Johnston G. C., Singer R. A. Prenylated isoforms of yeast casein kinase I, including the novel Yck3p, suppress the gcs1 blockage of cell proliferation from stationary phase. Mol Cell Biol. 1996 Oct;16(10):5375–5385. doi: 10.1128/mcb.16.10.5375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Whitney J. A., Gomez M., Sheff D., Kreis T. E., Mellman I. Cytoplasmic coat proteins involved in endosome function. Cell. 1995 Dec 1;83(5):703–713. doi: 10.1016/0092-8674(95)90183-3. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. Zanolari B., Raths S., Singer-Krüger B., Riezman H. Yeast pheromone receptor endocytosis and hyperphosphorylation are independent of G protein-mediated signal transduction. Cell. 1992 Nov 27;71(5):755–763. doi: 10.1016/0092-8674(92)90552-n. [DOI] [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES