The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins

Daiki Kawada, Hiromu Kobayashi, Tsuyoshi Tomita, Eisuke Nakata, Makoto Nagano, Daria Elisabeth Siekhaus, Junko Y. Toshima, Jiro Toshima

Research output: Contribution to journalArticle

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Abstract

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins.

Original languageEnglish
Pages (from-to)144-156
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1853
Issue number1
DOIs
Publication statusPublished - 2015 Jan 1

Fingerprint

GTPase-Activating Proteins
Membrane Proteins
Yeasts
Endosomes
Gene Deletion
Mating Factor
Membranes
Cell Membrane
Guanine Nucleotide Exchange Factors
Transport Vesicles
Pheromones
Vacuoles
Guanosine Triphosphate
GTP-Binding Proteins
Proteins
Hydrolysis

Keywords

  • Arf-GAP
  • Endocytosis
  • Intracellular trafficking
  • Small GTPase
  • Vesicular trafficking

ASJC Scopus subject areas

  • Cell Biology
  • Molecular Biology

Cite this

Kawada, D., Kobayashi, H., Tomita, T., Nakata, E., Nagano, M., Siekhaus, D. E., ... Toshima, J. (2015). The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins. Biochimica et Biophysica Acta - Molecular Cell Research, 1853(1), 144-156. https://doi.org/10.1016/j.bbamcr.2014.10.009

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins. / Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y.; Toshima, Jiro.

In: Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1853, No. 1, 01.01.2015, p. 144-156.

Research output: Contribution to journalArticle

Kawada, D, Kobayashi, H, Tomita, T, Nakata, E, Nagano, M, Siekhaus, DE, Toshima, JY & Toshima, J 2015, 'The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins', Biochimica et Biophysica Acta - Molecular Cell Research, vol. 1853, no. 1, pp. 144-156. https://doi.org/10.1016/j.bbamcr.2014.10.009
Kawada, Daiki ; Kobayashi, Hiromu ; Tomita, Tsuyoshi ; Nakata, Eisuke ; Nagano, Makoto ; Siekhaus, Daria Elisabeth ; Toshima, Junko Y. ; Toshima, Jiro. / The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins. In: Biochimica et Biophysica Acta - Molecular Cell Research. 2015 ; Vol. 1853, No. 1. pp. 144-156.
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