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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board: Buchner, Johannes / Lei, Ming / Ludwig, Stephan / Thomas, Douglas D. / Turk, Boris / Wittinghofer, Alfred


IMPACT FACTOR 2018: 3.014
5-year IMPACT FACTOR: 3.162

CiteScore 2018: 3.09

SCImago Journal Rank (SJR) 2018: 1.482
Source Normalized Impact per Paper (SNIP) 2018: 0.820

Online
ISSN
1437-4315
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Volume 386, Issue 2

Issues

Trafficking pathways of Cx49-GFP in living mammalian cells

Stephanie Breidert
  • Institute of Biophysics, University of Hannover, Herrenhäuserstr. 2, D-30419 Hannover, Germany
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/ Ralf Jacob
  • Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany and Department of Cytobiology, Philipps-University of Marburg, D-35037 Marburg, Germany
  • Other articles by this author:
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/ Anaclet Ngezahayo
  • Institute of Biophysics, University of Hannover, Herrenhäuserstr. 2, D-30419 Hannover, Germany
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/ Hans-Albert Kolb
  • Institute of Biophysics, University of Hannover, Herrenhäuserstr. 2, D-30419 Hannover, Germany
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/ Hassan Y. Naim
  • Department of Physiological Chemistry, School of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany
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Published Online: 2005-06-01 | DOI: https://doi.org/10.1515/BC.2005.019

Abstract

In the present study we examined the trafficking pathways of connexin49 (Cx49) fused to green fluorescent protein (GFP) in polar and non-polar cell lines. The Cx49 gene was isolated from ovine lens by RT-PCR. Cx49 cDNA was fused to GFP and the hybrid cDNA was transfected into several cell lines. After transfection of Cx49-GFP cDNA into HeLa cells, it was shown using the double whole-cell patch-clamp technique that the expressed fusion protein was still able to form conducting gap junction channels. Synthesis, assembly, and turnover of the Cx49-GFP hybrid protein were investigated using a pulse-chase protocol. A major 78-kDa protein band corresponding to Cx49-GFP could be detected with a turnover of 16–20 h and a half-life time of 10 h. The trafficking pathways of Cx49-GFP were monitored by confocal laser microscopy. Fusion proteins were localized in subcellular compartments, including the endoplasmic reticulum (ER), the ER-Golgi intermediate compartment, the Golgi apparatus, and the trans-Golgi network, as well as vesicles traveling towards the plasma membrane. Time-dependent sequential localization of Cx49-GFP in the ER and then the Golgi apparatus supports the notion of a slow turnover of Cx49-GFP compared to other connexins analyzed so far. Gap junction plaques resembling the usual punctuate distribution pattern could be demonstrated for COS-1 and MDCK cells. Basolateral distribution of Cx49-GFP was observed in polar MDCK cells, indicating specific sorting behavior of Cx49 in polarized cells. Together, this report describes the first characterization of biosynthesis and trafficking of lens Cx49.

Keywords: connexin49; green fluorescent protein (GFP); mammalian cells; protein assembly; protein transport; pulse-chase experiments

About the article

Corresponding author


Received: August 16, 2004

Accepted: November 26, 2004

Published Online: 2005-06-01

Published in Print: 2005-02-01


Citation Information: Biological Chemistry, Volume 386, Issue 2, Pages 155–160, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: https://doi.org/10.1515/BC.2005.019.

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