Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy

Sonoko Kawamura, Makoto Nagano, Junko Y. Toshima, Jiro Toshima

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to the cis- to medial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol in ric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to the trans-Golgi compartment and deletion of GYP6 increased the localization of Ypt6p at the trans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at the trans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary for intra-Golgi trafficking and protein recycling in the early Golgi compartment.

Original languageEnglish
Pages (from-to)519-525
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume450
Issue number1
DOIs
Publication statusPublished - 2014 Jul 18

Fingerprint

Recycling
Yeast
Yeasts
Vacuoles
Guanosine Triphosphate
Cytosol
Membranes
Homologous Recombination
Protein Transport
Cell membranes
Membrane Proteins
Proteins
Cell Membrane

Keywords

  • Budding yeast
  • N-terminal tagging
  • Rab/Ypt
  • Vesicle transport
  • Ypt6p

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy. / Kawamura, Sonoko; Nagano, Makoto; Toshima, Junko Y.; Toshima, Jiro.

In: Biochemical and Biophysical Research Communications, Vol. 450, No. 1, 18.07.2014, p. 519-525.

Research output: Contribution to journalArticle

@article{411ee8bc48a64060a08cf9b99bacb571,
title = "Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy",
abstract = "Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to the cis- to medial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol in ric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to the trans-Golgi compartment and deletion of GYP6 increased the localization of Ypt6p at the trans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at the trans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary for intra-Golgi trafficking and protein recycling in the early Golgi compartment.",
keywords = "Budding yeast, N-terminal tagging, Rab/Ypt, Vesicle transport, Ypt6p",
author = "Sonoko Kawamura and Makoto Nagano and Toshima, {Junko Y.} and Jiro Toshima",
year = "2014",
month = "7",
day = "18",
doi = "10.1016/j.bbrc.2014.06.002",
language = "English",
volume = "450",
pages = "519--525",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Academic Press Inc.",
number = "1",

}

TY - JOUR

T1 - Analysis of subcellular localization and function of the yeast Rab6 homologue, Ypt6p, using a novel amino-terminal tagging strategy

AU - Kawamura, Sonoko

AU - Nagano, Makoto

AU - Toshima, Junko Y.

AU - Toshima, Jiro

PY - 2014/7/18

Y1 - 2014/7/18

N2 - Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to the cis- to medial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol in ric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to the trans-Golgi compartment and deletion of GYP6 increased the localization of Ypt6p at the trans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at the trans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary for intra-Golgi trafficking and protein recycling in the early Golgi compartment.

AB - Ypt6p, the yeast homologue of mammalian Rab6, is involved in the multiple processes regulated by membrane trafficking such as vacuole maturation and membrane protein recycling. Although several lines of evidence suggest that Ypt6p is possibly localized to multiple membrane compartments, the precise localization of endogenous Ypt6p remains to be elucidated. In this study, we developed a novel method for N-terminal tagging of endogenous protein based on homologous recombination and investigated the subcellular localization and function of Ypt6p. Ypt6p and its GTP-bound form were predominantly localized to the cis- to medial-Golgi compartments whereas the GDP-bound form of Ypt6p was localized to the cytosol. Ric1p, a component of the specific GEF complex for Ypt6p, largely colocalized with Ypt6p in the early Golgi, and localization of Ypt6p changed to the cytosol in ric1Δ cells. On the other hand, Gyp6p, a putative GAP for Ypt6p, was localized to the trans-Golgi compartment and deletion of GYP6 increased the localization of Ypt6p at the trans-Golgi, suggesting that Gyp6p promotes the dissociation of Ypt6p from the Golgi when arriving at the trans-Golgi compartment. Additionally, we demonstrated that overexpression of the GDP-bound form of Ypt6p caused defective vacuole formation and recycling of Snc1p to the plasma membrane. These results suggest that the GTP-binding activity of Ypt6p is necessary for intra-Golgi trafficking and protein recycling in the early Golgi compartment.

KW - Budding yeast

KW - N-terminal tagging

KW - Rab/Ypt

KW - Vesicle transport

KW - Ypt6p

UR - http://www.scopus.com/inward/record.url?scp=84904736056&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84904736056&partnerID=8YFLogxK

U2 - 10.1016/j.bbrc.2014.06.002

DO - 10.1016/j.bbrc.2014.06.002

M3 - Article

VL - 450

SP - 519

EP - 525

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 1

ER -