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

The green fluorescent protein (GFP) of Aquorea victoria is fluorescent when expressed as a recombinant protein in eukaryotic cells and has b een used as a convenient marker of gene expression in vivo. It has als o been used as a marker of the intracellular targeting of recombinant fusion proteins (part GFP, part protein of interest) which have been t ransiently expressed in eukaryotic cells grown in tissue culture. Thus , the use of GFP has proved a useful tool to study intracellular event s in real-time. However, some transiently transfected cells fail to ex press, or localise correctly, the GFP-tagged protein. Therefore the pr oduction of stable cell lines expressing GFP-tagged integral membrane proteins may be essential for long-term studies. The generation of sta bly transfected eukaryotic cells expressing an integral membrane prote in with a known, but poorly characterised intracellular trafficking pa thway, would provide useful reagents for future, more precise, analysi s of that pathway. TGN38 is a type I integral membrane protein which c ycles between the trans-Golgi network (TGN) and cell surface; at stead y state it is localised to the TGN. As such, TGN38 is an ideal candida te for tagging with GFP. We have generated cDNA constructs encoding ra tTGN38 tagged at either the N- or C terminus with GFP. Transiently tra nsfected rat (NRK) cells expressed active fluorophore, but failed to s how correct localisation of the fusion protein. In contrast, both cons tructs are appropriately localised in stably transfected NRK cells and both are fluorescent. Furthermore, the recombinant GFP-tagged protein s and the endogenous TGN38 molecules show identical responses to drugs and temperature blocks known to perturb intracellular morphology and membrane traffic pathways. In fact morphological changes to the TGN in duced by brefeldin A were observed at earlier time points than had bee n described previously using immunofluorescence analysis of fixed cell s, thus validating the use of in vivo, real-time analysis of GFP-tagge d proteins. In addition, we show that (in contrast to the situation in COS cells) elevated expression of ratTGN38 in NRK cells does not lead to a fragmentation of the TGN; this has implications for the role whi ch TGN38 is playing in the maintenance of the morphology of the TGN. T he data we present demonstrate that: (i) it is possible to generate st able cell lines expressing integral membrane proteins tagged with GFP; (ii) the GFP tag remains fluorescent when expressed on either the cyt osolic or the lumenal side of all membranes of the secretory pathway u p to and including that of the TGN; (iii) the GFP tag does not interfe re with the transport of TGN38 along the secretory pathway or its rete ntion in the TGN; (iv) GFP remains fluorescent in cells which have bee n processed for immunofluorescence analysis (using either paraformalde hyde or methanol fixation); and (v) TGN38 plays a role in maintaining the morphology of the TGN. Thus, stably transfected cells expressing G FP-tagged integral membrane proteins can be used as effective tools fo r the real-time study of intracellular morphology and membrane traffic pathways in eukaryotic cells.