Details
| Original language | English |
|---|---|
| Pages (from-to) | 155-60 |
| Number of pages | 6 |
| Journal | Biological chemistry |
| Volume | 386 |
| Issue number | 2 |
| Publication status | Published - Feb 2005 |
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
- Animals, Biological Transport, COS Cells, Cell Line, Cell Polarity, Connexins/metabolism, Dogs, Gap Junctions/metabolism, Green Fluorescent Proteins/metabolism, HeLa Cells, Humans, Kidney, Microscopy, Confocal, Recombinant Fusion Proteins/metabolism
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In: Biological chemistry, Vol. 386, No. 2, 02.2005, p. 155-60.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Trafficking pathways of Cx49-GFP in living mammalian cells
AU - Breidert, Stephanie
AU - Jacob, Ralf
AU - Ngezahayo, Anaclet
AU - Kolb, Hans-Albert
AU - Naim, Hassan Y
N1 - Funding information: The authors would like to thank Dr. Rüdiger Junker for preparation of ovine lenses and Prof. Arne Ernst for helpful discussions. This work was supported by the Fritz Thyssen-Stiftung.
PY - 2005/2
Y1 - 2005/2
N2 - 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.
AB - 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.
KW - Animals
KW - Biological Transport
KW - COS Cells
KW - Cell Line
KW - Cell Polarity
KW - Connexins/metabolism
KW - Dogs
KW - Gap Junctions/metabolism
KW - Green Fluorescent Proteins/metabolism
KW - HeLa Cells
KW - Humans
KW - Kidney
KW - Microscopy, Confocal
KW - Recombinant Fusion Proteins/metabolism
U2 - 10.1515/BC.2005.019
DO - 10.1515/BC.2005.019
M3 - Article
C2 - 15843159
VL - 386
SP - 155
EP - 160
JO - Biological chemistry
JF - Biological chemistry
SN - 1431-6730
IS - 2
ER -