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The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20 degrees C) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.

Interferons (IFNs) are secretory proteins consisting of type I (a, b, w, etc.) and type II (g) IFN, which exhibit a wide range of biological activities.2,3) IFNs have been used clinically as antiviral and antitumor reagents, 4,5) and IFN gene transfer has also been attempted for therapeutic purposes. 6,7) In regularly used non-polarized cells such as fibroblasts, exogenoulsy synthesized IFNs will be transported through organella, including the endoplasmic reticulum (ER), the Golgi and the trans-Golgi network (TGN), and secreted around the cells. In contrast, IFN expression in polarized epithelial cells involves specific issues about the direction of its secretion (secretion polarity) and regulation of the vectorial protein sorting in cells, because the cell surface of polarized epithelial cells is divided into apical and basolateral domains with distinct membrane protein and lipid compositions. 8,9) Secretory proteins such as IFNs are likely to be transported in the same manner as membrane proteins. 10)We have investigated the secretion polarity of IFN-b exogenously expressed in several epithelial cell monolayers and found that IFN-b transiently expressed by gene transfection was predominantly secreted from the cell membrane side on which the transfection has been carried out, whereas the secretion of constitutive IFN-b from stable transformants was apparently unpolarized in Pam-T and Madin-Darby canine kidney (MDCK) type I cells.11,12) Similar phenomena have been observed in IFN production by type I IFN inducer, polyriboinosinic acid : polyribocytidylic acid (poly I : poly C), in various epithelial cell monolayers.13) Furthermore, subcellular localization analysis using enhanced green fluorescent protein (EGFP)-tagged IFN-b had shown that identical IFNb was conveyed to the apical and/or basolateral membrane side, depending on the gene expression strategy, presumably by post-TGN additional sorting events.12) On the other hand, the gene expression mode-dependent secretion polarity in stable and transient expression of IFN-b was not found in LLC-PK1 and MDCK type II cells.14) For another protein, stably expressed human erythropoietin showed apical preferential secretion although polarized secretion was not observed in transiently expressed erythropoietin in MDCK type II cells.15) These data suggested that the gene expression mode-dependent secretion polarity depends on the cell types and property of proteins itself.It has been known that regions of the plasma membrane contain microdomains, called detergent-resistant membranes (DRMs) or lipid rafts, 16) that are rich in glycosphingolipids and cholesterol, 17) which include or exclude selected proteins and are believed to play important roles in protein trafficking and signal transduction. 18,19) In polarized epithelial cells, particular proteins associated with lipid rafts/DRMs, such as the glycosylphosphatidylinositol (GPI)-anchored proteins, 20,21) the Myelin and lymphocyte protein (MAL) 22,23) and caveolin-1, 24) are important for apical transport of membrane p...

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