Department of Life Science and National Research Laboratory, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea1
Author for correspondence: Woo Jin Park. Fax +82 62 970 2484. e-mail wjpark{at}kjist.ac.kr
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Abstract |
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In this study, we have determined the substrate specificity of the NIa protease by utilizing a recently developed screening method referred to as GASP (genetic assay for site-specific proteolysis) (Hawkins et al., 1999 ; Steiner et al., 1999
; Kim et al., 2000
; Kang et al., 2001
). The principle of GASP is shown in Fig. 1(a
). A fusion protein, SteSubLex, was generated in which a transcription factor, LexA-b42, is linked to the truncated cytoplasmic domain of an integral membrane protein, STE2, by a short linker containing a substrate sequence of the NIa protease. The rationale behind this approach was that: (i) in the absence of NIa protease activity, LexA-b42 remains anchored to the cytoplasmic membrane (left panel); (ii) in the presence of the NIa protease, the substrate sequence is cleaved, resulting in the release of LexA-b42, which can in turn enter the nucleus and activate reporter genes (right panel).
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Good substrate sequences for the NIa protease were selected basically as described previously (Kim et al., 2000 ). Briefly, we incorporated randomization into the P6-P5-P4, P3-P2 and P1-P1' positions of the 6K1/CI junction in separate experiments. Each randomized sequence was designed to contain any of the 20 natural amino acids. Yeast cells harbouring pGAL-NIa were transformed with plasmid DNA of these randomized substrate libraries and then plated onto the plates lacking Trp and His (Trp1 and His3 are the marker genes for the substrate and protease vectors, respectively), and then replica-plated to selective plates lacking Trp, His and Leu (Leu2 is the reporter gene that is activated by LexA-b42) and containing either glucose or galactose. They were incubated for 34 days at 30 °C. Colonies that grew on selective plates containing galactose but not on plates containing glucose were assumed to harbour substrate sequences that were specifically cleaved by the NIa protease. To select substrate sequences which were cleaved as efficiently as the 6K1/CI junction site, colonies that grew as fast as transformants harbouring pADH-SteSubLex and pGAL-NIa were selected. These colonies were further tested on X-Gal plates and only strong blue colonies were further characterized (lacZ is another reporter gene that is activated by LexA-b42). The substrate regions of the selected positive colonies were amplified by PCR and directly subjected to nucleotide sequencing. The results are shown in Fig. 2
, and the deduced consensus sequence is shown in Fig. 3
. Val, His and Gln were the most frequently occurring residues at positions P4, P2 and P1, respectively. This result is consistent with the fact that these amino acids are the most highly conserved residues in the cleavage junctions of the TuMV polyprotein (Nicolas & Laliberté, 1992
; Yoon et al., 2000
). In particular, His appeared to be strictly required at position P2 for efficient cleavage. Previous studies have shown that Gln at P1 is essential for the cleavage by tobacco etch virus NIa proteases (Dougherty et al., 1988
). Notably, the type of amino acid at position P1 was less strict according to results from our experiments. Considering that some of the cleavage sites of TuMV do not have Gln at P1 and they are less efficiently cleaved (Kim et al., 1996
), it is likely that a small number of suboptimal sequences were selected during screening. Ser or Ile were the two most favoured residues at position P1'. Arg was the most frequently occurring residue at P3, although eight other amino acids were also found at this position. Aliphatic amino acids (Gly, Ala, Leu, Ile) were favoured at P5. No obvious preference was observed at position P6. Taken together, we suggest that Yaa-Val-Arg-His-Gln
Ser is the most favourable cleavage sequence for the NIa protease, where Yaa is an aliphatic amino acid and the scissile bond is located between Gln and Ser (Fig. 3
).
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References |
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Estojak, J., Brent, R. & Golemis, E. A. (1995). Correlation of two-hybrid affinity data with in vitro measurements. Molecular and Cellular Biology 15, 5820-5829.[Abstract]
Hawkins, C. J., Wang, S. L. & Hay, B. A. (1999). A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1. Proceedings of the National Academy of Sciences, USA 96, 2885-2890.
Kang, H., Kim, S. Y. & Park, W. J. (2001). An improved strategy for a genetic assay for site-specific proteolysis. Molecules and Cells 11, 263-266.[Medline]
Kim, D. H., Hwang, D. C., Kang, B. H., Lew, J., Han, J., Song, B. D. & Choi, K. Y. (1996). Effects of internal cleavages and mutations in the C-terminal region of NIa protease of turnip mosaic potyvirus on the catalytic activity. Virology 226, 183-190.[Medline]
Kim, S. Y., Park, K. W., Lee, Y. J., Back, S. H., Goo, J. H., Park, O. K., Jang, S. K. & Park, W. J. (2000). In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis. Analytical Biochemistry 284, 42-48.[Medline]
Lee, Y. J., Kang, H., Rho, S. H., Eom, S. H. & Park, W. J. (2001). Assessment of substrate specificity of hepatitis G virus NS3 protease by a genetic method. Biochemical and Biophysical Research Communications 286, 171175.[Medline]
Nicolas, O. & Laliberté, J.-F. (1992). The complete nucleotide sequence of turnip mosaic potyvirus RNA. Journal of General Virology 73, 2785-2793.[Abstract]
Riechmann, J. L., Laín, S. & García, J. A. (1992). Highlights and prospects of potyvirus molecular biology. Journal of General Virology 73, 1-16.[Medline]
Shattuck, V. I., Brolley, B., Stobbs, L. W. & Lougheed, E. C. (1989). The effect of turnip mosaic virus infection on the mineral content and storability of field-grown rutabaga. Communications in Soil Science and Plant Analysis 20, 581-595.
Shukla, D. D., Ward, C. W. & Brunt, A. A. (1994). The Potyviridae. Wallingford: CAB International.
Steiner, H., Pesold, B. & Haass, C. (1999). An in vivo assay for the identification of target proteases which cleave membrane-associated substrates. FEBS Letters 463, 245-249.[Medline]
Yoon, H. Y., Choi, K. Y. & Song, B. D. (2000). Fluorometric assay of turnip mosaic virus NIa protease. Analytical Biochemistry 277, 228-231.[Medline]
Received 20 June 2001;
accepted 17 August 2001.