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Title: Glycosylatable GFP as a compartment-specific membrane topology reporter

Abstract

Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.

Authors:
;  [1];  [2];  [3];  [1]
  1. School of Biological Sciences, Seoul National University, Seoul 151-747 (Korea, Republic of)
  2. Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm (Sweden)
  3. (Sweden)
Publication Date:
OSTI Identifier:
22210318
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 427; Journal Issue: 4; Other Information: Copyright (c) 2012 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ENDOPLASMIC RETICULUM; FLUORESCENCE; MEMBRANE PROTEINS; MEMBRANES; SACCHAROMYCES CEREVISIAE

Citation Formats

Lee, Hunsang, Min, Jisoo, Heijne, Gunnar von, Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna, and Kim, Hyun, E-mail: joy@snu.ac.kr. Glycosylatable GFP as a compartment-specific membrane topology reporter. United States: N. p., 2012. Web. doi:10.1016/J.BBRC.2012.09.138.
Lee, Hunsang, Min, Jisoo, Heijne, Gunnar von, Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna, & Kim, Hyun, E-mail: joy@snu.ac.kr. Glycosylatable GFP as a compartment-specific membrane topology reporter. United States. doi:10.1016/J.BBRC.2012.09.138.
Lee, Hunsang, Min, Jisoo, Heijne, Gunnar von, Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna, and Kim, Hyun, E-mail: joy@snu.ac.kr. Fri . "Glycosylatable GFP as a compartment-specific membrane topology reporter". United States. doi:10.1016/J.BBRC.2012.09.138.
@article{osti_22210318,
title = {Glycosylatable GFP as a compartment-specific membrane topology reporter},
author = {Lee, Hunsang and Min, Jisoo and Heijne, Gunnar von and Science for Life Laboratory, Stockholm University, Box 1031, SE-171 21 Solna and Kim, Hyun, E-mail: joy@snu.ac.kr},
abstractNote = {Highlights: Black-Right-Pointing-Pointer An N-linked glycosylation site is introduced near the GFP fluorophore. Black-Right-Pointing-Pointer gGFP is not glycosylated and is fully fluorescent in the cytosol. Black-Right-Pointing-Pointer gGFP is glycosylated and non-fluorescent in the lumen of the ER. Black-Right-Pointing-Pointer gGFP is fused to membrane proteins of known topology. Black-Right-Pointing-Pointer Its applicability as a membrane topology reporter is demonstrated. -- Abstract: Determination of the membrane topology is an essential step in structural and functional studies of integral membrane proteins, yet the choices of membrane topology reporters are limited and the experimental analysis can be laborious, especially in eukaryotic cells. Here, we present a robust membrane topology reporter, glycosylatable green fluorescent protein (gGFP). gGFP is fully fluorescent in the yeast cytosol but becomes glycosylated and does not fluoresce in the lumen of the endoplasmic reticulum (ER). Thus, by assaying fluorescence and the glycosylation status of C-terminal fusions of gGFP to target membrane proteins in whole-cell lysates, the localization of the gGFP moiety (and hence the fusion joint) relative to the ER membrane can be unambiguously determined.},
doi = {10.1016/J.BBRC.2012.09.138},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 4,
volume = 427,
place = {United States},
year = {2012},
month = {11}
}