Proteolytic activity assayed by subcellular localization switching of a substrate

Abstract

An approach to assay proteolytic activity in vivo by altering the subcellular localization of a labelled substrate was demonstrated. The assay included a protein shuttling between different cellular compartments and a site-specific recombinant protease. The shuttle protein used was the human immunodeficiency virus type 1 (HIV-1) Rev protein tandemly fused to the enhanced green fluorescent protein (EGFP) and the red fluorescent protein (RFP), while the protease was the site-specific protease VP24 from the herpes simplex virus type 1 (HSV-1). The fluorescent proteins in the Rev fusion protein were separated by a cleavage site specific for the VP24 protease. When co-expressed in COS-7 cells proteolysis was observed by fluorescence microscopy as a shift from a predominantly cytoplasmic localization of the fusion protein RevEGFP to a nuclear localization while the RFP part of the fusion protein remained in the cytoplasm. The cleavage of the fusion protein by VP24 was confirmed by Western blot analysis. The activity of VP24, when tagged N-terminally by the Myc-epitope, was found to be comparable to VP24. These results demonstrates that the activity and localization of a recombinantly expressed protease can be assessed by protease-mediated cleavage of fusion proteins containing a specific protease cleavage site.

Keywords: CLSM, confocal laser scanning microscopy; EGFP, enhanced green fluorescent protein; Green fluorescent protein; HIV, human immunodeficiency virus type 1; HIV-1 Rev; HSV-1 protease; HSV-1, herpes simplex virus type 1; HTLV-1, human T-cell leukaemia virus type 1; NES, nuclear export signal; NLS, nuclear localization signal; NOS, nucleolar localization signal; RFP, red fluorescent protein; Red fluorescent protein.

Fig. 1

Schematic representations of Rev fusion proteins, protease fusion protein and localization of these constructs in COS-7 cells. (A) RevEGFPasDsRed1consists of Rev, enhanced green fluorescent protein (EGFP) and red fluorescent protein (DsRed1). Between the EGFP and DsRed1 the HSV-1 VP24 recognition sequence AEAGALVNASSAAHVDV was inserted. RevEGFP-DsRed1 represents the fusion protein without the HSV-1 optimal recognition sequence while RevEGFP is the fusion protein without DsRed1. The HSV-1 VP24 protein is N-terminally tagged with the Myc epitope. The molecular sizes are indicated. (B) The intracellular steady state localization of RevEGFPasDsRed1, RevEGFP-DsRed1, RevEGFP and MycVP24 in COS-7 cells. The anti-Myc Mab 9E10 was used for immunofluorescent detection of MycVP24. The cells were fixed 48 h post-transfection and analysed by confocal laser scanning microscopy (CSLM).

Fig. 2

Protease activity assayed by changes in localization of RevEGFPasDsRed1 after transfection with pRevGasR without or with pcMycVP24 in COS-7 cells. Panels A–C show cells after transfection with pRevGasR, panels D-F show cells after cotransfection with pRevGasR and pcMycVP24. The left, middle and right columns show images in red, green and merged channels, respectively. The cells were analysed by confocal laser scanning microscopy (CSLM). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Activity of HSV-1 VP24. COS-7 cells were transfected with the plasmids indicated above the lanes. A) Western blot analysis of COS-7 cells expressing RevEGFPasDsRed1 protein in the presence (+) or absence (−) of VP24 protease. B) Western blot analysis of RevEGFP-DsRed1 proteins in the presence (+) or absence (-) of VP24. C) Western blot analysis of RevEGFPasDsRed1 protein in the presence (+) or absence (−) of MycVP24. The Rev fusion proteins were detected using the anti-Rev Mab 8E7. The RevEGFP in lane 3 is shown as a molecular size reference. Arrowheads: 71-kDa Rev fusion protein and the 45-kDa cleavage product.