Trafficking pathways of Cx49-GFP in living mammalian cells.
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.
Breidert
Stephanie
S
Institute of Biophysics, University of Hannover, Herrenhäuserstrasse 2, D-30419 Hannover, Germany.
Jacob
Ralf
R
Ngezahayo
Anaclet
A
Kolb
Hans-Albert
HA
Naim
Hassan Y
HY
D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002460:Cell Line; D016764:Cell Polarity; D017630:Connexins / Q000378:metabolism*; D004285:Dogs; D017629:Gap Junctions / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D018613:Microscopy, Confocal; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D017630:Connexins; D011993:Recombinant Fusion Proteins; C109089:connexin 49 protein, Ovis aries; D049452:Green Fluorescent Proteins