TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible role for ratTGN38

摘要: The green fluorescent protein (GFP) of Aquorea victoria is when expressed as a recombinant in eukaryotic cells and has been used convenient marker gene expression vivo. It also the intracellular targeting fusion proteins (part GFP, part interest) which have transiently grown tissue culture. Thus, use GFP proved useful tool to study events real-time. However, some transfected fail express, or localise correctly, GFP-tagged protein. Therefore production stable cell lines expressing integral membrane may be essential for long-term studies. generation stably an with known, but poorly characterised trafficking pathway, would provide reagents future, more precise, analysis that pathway. TGN38 type I cycles between trans-Golgi network (TGN) surface; at steady state it localised TGN. As such, ideal candidate tagging GFP. We generated cDNA constructs encoding ratTGN38 tagged either N- C terminus Transiently rat (NRK) active fluorophore, failed show correct localisation In contrast, both are appropriately NRK fluorescent. Furthermore, endogenous molecules identical responses drugs temperature blocks known perturb morphology traffic pathways. fact morphological changes TGN induced by brefeldin A were observed earlier time points than had described previously using immunofluorescence fixed cells, thus validating vivo, real-time proteins. addition, we (in contrast situation COS cells) elevated does not lead fragmentation TGN; this implications role playing maintenance data present demonstrate that: (i) possible generate GFP; (ii) tag remains on cytosolic lumenal side all membranes secretory pathway up including (iii) interfere transport along its retention (iv) processed (using paraformaldehyde methanol fixation); (v) plays maintaining can effective tools pathways cells.