Journal of Cell Science, Vol 109, Issue 12 2915-2926, Copyright © 1996 by Company of Biologists

JOURNAL ARTICLES

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

M Girotti and G Banting
Department of Biochemistry, University of Bristol, UK.

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

This article has been cited by other articles:

				R. Rollason, V. Korolchuk, C. Hamilton, P. Schu, and G. Banting Clathrin-mediated endocytosis of a lipid-raft-associated protein is mediated through a dual tyrosine motif J. Cell Sci., November 1, 2007; 120(21): 3850 - 3858. [Abstract] [Full Text] [PDF]

				V. Jakob, A. Schreiner, R. Tikkanen, and A. Starzinski-Powitz Targeting of Transmembrane Protein Shrew-1 to Adherens Junctions Is Controlled by Cytoplasmic Sorting Motifs Mol. Biol. Cell, August 1, 2006; 17(8): 3397 - 3408. [Abstract] [Full Text] [PDF]

				R. Giau, J. Carrette, J. Bockaert, and V. Homburger Constitutive Secretion of Protease Nexin-1 by Glial Cells and Its Regulation by G-Protein-Coupled Receptors J. Neurosci., September 28, 2005; 25(39): 8995 - 9004. [Abstract] [Full Text] [PDF]

				A. Kyttala, K. Yliannala, P. Schu, A. Jalanko, and J. P. Luzio AP-1 and AP-3 Facilitate Lysosomal Targeting of Batten Disease Protein CLN3 via Its Dileucine Motif J. Biol. Chem., March 18, 2005; 280(11): 10277 - 10283. [Abstract] [Full Text] [PDF]

				A. Kyttala, G. Ihrke, J. Vesa, M. J. Schell, and J. P. Luzio Two Motifs Target Batten Disease Protein CLN3 to Lysosomes in Transfected Nonneuronal and Neuronal Cells Mol. Biol. Cell, March 1, 2004; 15(3): 1313 - 1323. [Abstract] [Full Text] [PDF]

				G. Blot, K. Janvier, S. Le Panse, R. Benarous, and C. Berlioz-Torrent Targeting of the Human Immunodeficiency Virus Type 1 Envelope to the trans-Golgi Network through Binding to TIP47 Is Required for Env Incorporation into Virions and Infectivity J. Virol., June 15, 2003; 77(12): 6931 - 6945. [Abstract] [Full Text] [PDF]

				S. Lee, B. Park, and K. Ahn Determinant for Endoplasmic Reticulum Retention in the Luminal Domain of the Human Cytomegalovirus US3 Glycoprotein J. Virol., February 1, 2003; 77(3): 2147 - 2156. [Abstract] [Full Text] [PDF]

				V. Sytnyk, I. Leshchyns'ka, M. Delling, G. Dityateva, A. Dityatev, and M. Schachner Neural cell adhesion molecule promotes accumulation of TGN organelles at sites of neuron-to-neuron contacts J. Cell Biol., November 25, 2002; 159(4): 649 - 661. [Abstract] [Full Text] [PDF]

				B. A. Rous, B. J. Reaves, G. Ihrke, J. A.G. Briggs, S. R. Gray, D. J. Stephens, G. Banting, and J. P. Luzio Role of Adaptor Complex AP-3 in Targeting Wild-Type and Mutated CD63 to Lysosomes Mol. Biol. Cell, March 1, 2002; 13(3): 1071 - 1082. [Abstract] [Full Text] [PDF]

				D. Stephens and R Pepperkok Illuminating the secretory pathway: when do we need vesicles? J. Cell Sci., January 3, 2001; 114(6): 1053 - 1059. [Abstract] [PDF]

				A. Dodd, P. M. Curtis, L. C. Williams, and D. R. Love Zebrafish: bridging the gap between development and disease Hum. Mol. Genet., October 1, 2000; 9(16): 2443 - 2449. [Abstract] [Full Text] [PDF]

				B. Reaves, P. Row, N. Bright, J. Luzio, and H. Davidson Loss of cation-independent mannose 6-phosphate receptor expression promotes the accumulation of lysobisphosphatidic acid in multilamellar bodies J. Cell Sci., January 11, 2000; 113(22): 4099 - 4108. [Abstract] [PDF]

				J. B. McCabe and L. G. Berthiaume Functional Roles for Fatty Acylated Amino-terminal Domains in Subcellular Localization Mol. Biol. Cell, November 1, 1999; 10(11): 3771 - 3786. [Abstract] [Full Text]

				J. Hirst, N. A. Bright, B. Rous, and M. S. Robinson Characterization of a Fourth Adaptor-related Protein Complex Mol. Biol. Cell, August 1, 1999; 10(8): 2787 - 2802. [Abstract] [Full Text]

				P. L. Ferguson and W. F. Flintoff Topological and Functional Analysis of the Human Reduced Folate Carrier by Hemagglutinin Epitope Insertion J. Biol. Chem., June 4, 1999; 274(23): 16269 - 16278. [Abstract] [Full Text] [PDF]

				S. Nelson, R. D. Horvat, J. Malvey, D. A. Roess, B. G. Barisas, and C. M. Clay Characterization of an Intrinsically Fluorescent Gonadotropin-Releasing Hormone Receptor and Effects of Ligand Binding on Receptor Lateral Diffusion Endocrinology, February 1, 1999; 140(2): 950 - 957. [Abstract] [Full Text]

				C. M. Sanderson and G. L. Smith Vaccinia Virus Induces Ca2+-Independent Cell-Matrix Adhesion during the Motile Phase of Infection J. Virol., December 1, 1998; 72(12): 9924 - 9933. [Abstract] [Full Text] [PDF]

				J. Llopis, J. M. McCaffery, A. Miyawaki, M. G. Farquhar, and R. Y. Tsien Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins PNAS, June 9, 1998; 95(12): 6803 - 6808. [Abstract] [Full Text] [PDF]

				B. J. Reaves, G. Banting, and J. P. Luzio Lumenal and Transmembrane Domains Play a Role in Sorting Type I Membrane Proteins on Endocytic Pathways Mol. Biol. Cell, May 1, 1998; 9(5): 1107 - 1122. [Abstract] [Full Text]

				T. Nakata, S. Terada, and N. Hirokawa Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons J. Cell Biol., February 9, 1998; 140(3): 659 - 674. [Abstract] [Full Text] [PDF]

				G Banting, R Maile, and E. Roquemore The steady state distribution of humTGN46 is not significantly altered in cells defective in clathrin-mediated endocytosis J. Cell Sci., January 12, 1998; 111(23): 3451 - 3458. [Abstract] [PDF]

				A. A. Peden, R. E. Rudge, W. W.Y. Lui, and M. S. Robinson Assembly and function of AP-3 complexes in cells expressing mutant subunits J. Cell Biol., January 21, 2002; 156(2): 327 - 336. [Abstract] [Full Text] [PDF]