A genetic system for detection of protein nuclear import and export

Abstract

We have developed a simple genetic assay to detect active nuclear localization (NLS) and export signals (NES) on the basis of their function within yeast cells. The bacterial LexA protein was modified (mLexA) to abolish its intrinsic NLS and fused to the activation domain of the yeast Gal4p (Gal4AD) with or without the SV40 large T-antigen NLS. In the import assay, if a tested protein fused to mLexA-Gal4AD contains a functional NLS, it will enter the cell nucleus and activate the reporter gene expression. In the export assay, if a tested protein fused to mLexA-SV40 NLS-Gal4AD contains a functional NES, it will exit into the cytoplasm, decreasing the reporter gene expression. We tested this system with known NLS and NES and then used it to demonstrate a NES activity of the capsid protein of a plant geminivirus. This approach may help to identify, analyze, and select for proteins containing functional NLS and NES.

Nuclear Shuttling of She2p Couples ASH1 mRNA Localization to its Translational Repression by Recruiting Loc1p and Puf6p

“…It was possible that the She2p-mLexA-GAL4AD fusion protein could diffuse passively through the nuclear pores. To eliminate this possibility, the 70-kDa protein VirE2, from Agrobacterium tumefaciens, was added to the She2p-mLexA-GAL4AD in order to increase the size of the fusion protein (Rhee et al, 2000). Even this large fusion protein was actively imported in the nucleus in a She2p-dependent manner (Figure 1A), suggesting that She2p contains an active NLS.…”

“…However, because the functional structure of She2p is that of a homodimer of more than 50 kDa (Niessing et al, 2004), it is possible that active nuclear import is required. To determine if She2p shuttles between the nucleus and the cytoplasm actively or passively, a yeast genetic assay was used (Rhee et al, 2000). In this assay, the protein of interest is fused to a chimera made of a modified LexA protein (mLexA), containing a disrupted NLS, and of the GAL4 activation domain (mLexA-GAL4AD).…”

Characterization of nuclear localization signals in the type III effectors HsvG and HsvB of the gall-forming bacterium Pantoea agglomerans

“…The strategy of the pNIA assay is based on expression in yeast cells of a triple fusion that involves a protein comprising bacterial mLexA, the yeast Gal4p activation domain (Gal4AD), and the tested protein. The mLexA-Gal4AD chimeric protein is unable to localize to the nucleus, because the NLS in mLexA has been eliminated, and because of this disability the transcriptional activator Gal4AD cannot activate the mLexA operon in yeast cells, which results in arrest of yeast-cell growth on histidine-deficient medium (Rhee et al, 2000). Only when the test protein fused to the mLexA-Gal4AD chimera possesses a functional NLS will the mLexA direct the mLexA operator into yeast nuclei and enable yeast-cell growth on histidine-deficient medium (Rhee et al, 2000;Tzfira & Citovsky, 2001).…”

“…The mLexA-Gal4AD chimeric protein is unable to localize to the nucleus, because the NLS in mLexA has been eliminated, and because of this disability the transcriptional activator Gal4AD cannot activate the mLexA operon in yeast cells, which results in arrest of yeast-cell growth on histidine-deficient medium (Rhee et al, 2000). Only when the test protein fused to the mLexA-Gal4AD chimera possesses a functional NLS will the mLexA direct the mLexA operator into yeast nuclei and enable yeast-cell growth on histidine-deficient medium (Rhee et al, 2000;Tzfira & Citovsky, 2001). However, pNIA encodes a small protein that lacks NLS sequences which may enter the nucleus by simple diffusion and therefore cannot be used in a nuclear localization assay for small proteins or peptides.…”

“…The nuclear import assay of the putative NLSs of HsvG was performed with pNIA vector, according to Rhee et al (2000). The strategy of the pNIA assay is based on expression in yeast cells of a triple fusion that involves a protein comprising bacterial mLexA, the yeast Gal4p activation domain (Gal4AD), and the tested protein.…”