Nuclear protein import assays

Ever since the demonstration that synthetic peptides could target a protein to the nucleus when cross-linked to it (Goldfarb et ai, 1986), peptide-protein conjugates have been used widely in nuclear protein import assays in vivo and in vitro. The method described here is for the preparation of a fluorescent bovine serum albumin, SV40 NLS conjugate. However, this basic protocol can be adapted easily to allow the conjugation to other proteins, such as the naturally fluorescent protein allophycocyanin (Adam etai, 1991). 

We thank lain Mattaj for advice and support, Paul Fisher for the rapid immunofluorescence method, and Dirk Gdrlich for sharing the latest improvements to the nuclear protein import assay. We particularly thank Rainer Saffrich for constant help and advice on quantitative fluorescence microscopy. Colin Dingwall acknowledges the financial support of the Council for Tobacco Research. 

The following method of cell permeabilization is a modification of the method originally described by Adam et al. (1990). We have successfully applied it to a number of different cell types to give permeabilized cells competent for nuclear protein and snRNP import in vitro. It has the advantage that large numbers of permeabilized cells can be produced in a single batch and stored frozen in small aliquots for transport experiments. This eliminates the variation that can arise when cells are permeabilized on cover slips and each cover slip is then used for a single data point in an assay. 

Most current assays use immunofluorescence or GFP fluorescence to evaluate the nucleocytoplasmic distribution of endogenous nuclear proteins (Corbett et al., 1995 Lim et al, 1995 Schlenstedt et al, 1995) or NLS-containing reporter proteins (Nehrbass et al, 1993 Schlaich and Hurt, 1995) in the steady state. These methods are usually sensitive only to defects that severely impair the transport apparatus (see example below). Even the use of inducible promoters to express reporter proteins after shifting cells to nonpermissive conditions (Corbett et al, 1995 Schlenstedt et al, 1995) requires at least 30-60 min to produce an adequate signal and is, therefore, often too slow. A clever assay was recently described to measure defects in nuclear export signal-mediated protein export in yeast (Lee et al, 1996). An attractive in vivo approach now in use in tissue culture cells is the application of hormone-stimulated GFP-glucocorticoid receptor import to study import kinetics (Htun et al, 1996 Carey et al, 1996). 

Assay developments for important target classes such as protein kinases and phosphatases, proteases, nuclear receptors, G protein-coupled receptors, ion channels, and heat shock proteins... 

In some experiments concerned with the mechanism by which tryptophan acted to improve hepatic protein synthesis after toxic injury, the ability of tryptophan to stimulate hepatic mRNA synthesis, nucleocytoplasmic translocation of RNA in vitro, and nuclear envelope nucleoside triphosphatase activity after hepatotoxic injury was measured.188 Nucleoside triphosphatase (Mg2+-dependent adenosine triphosphatase, EC 3.6.1.3.1) was assayed since it is present in mammalian liver nuclear envelopes,224 and there is evidence that this enzyme is involved in nucleocytoplasmic translocation of RNA.221 All of these parameters were elevated significantly by tryptophan after agents such as actinomycin D, cordycepin, ethionine, puromycin, and hypertonic NaCl demonstrated a curative effect by tryptophan, but not after tryptophan following CC14, NaF, and sparsomycin demonstrated no improvement with tryptophan. These findings emphasized the importance of the role that tryptophan plays in stimulating the availability of cytoplasmic... 

Early work on the mechanism of nuclear import focused on proteins containing a basic NLS, similar to the one in SV40 large T-antlgen. A digitonin-permeabilized cell system provided an in vitro assay for analyzing soluble cytosolic components required for nuclear Import (Figure... 

Many inflammatory cytokines including IL-8 are regulated at transcriptional levels, and a variety of transcription factors such as nuclear factor-K B (NFkB) and activator protein-1 (AP-1) play important roles in such processes. Abe and coworkers [71] demonstrated that CAM repressed TNF-a-induced AP-1 activation in human bronchial epithelial cells. We studied the effect of EM and CAM on the phorbol myristate acetate (PMA)-induced activation of NFkB and AP-1. Pretreatment of EM and CAM before the PMA treatment showed an inhibitory effect on both of the transcription factors as assessed by electrophoretic mobility shift assay (EMSA) (Fig. 14) [20]. In contrast, the macrolides showed no effect on the activation of cyclic AMP-responsive element binding protein (CREB), suggesting that the suppressive effect on some transcription factors is specific. We further evaluated the effect of EM on the phosphorylation of inhibitor of NFkB (IkB), which is a crucial step for transactivation of NFkB. EM did not influence the phosphorylation processes in vitro (Okazaki et at, unpublished data, January 2001). These data suggest that EM acts at the process of nuclear translocation of... 

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