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Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor - PubMed

  • ️Fri Jan 01 2010

Three non-autonomous signals collaborate for nuclear targeting of CrMYC2, a Catharanthus roseus bHLH transcription factor

Sabah Hedhili et al. BMC Res Notes. 2010.

Abstract

Background: CrMYC2 is an early jasmonate-responsive bHLH transcription factor involved in the regulation of the expression of the genes of the terpenic indole alkaloid biosynthesis pathway in Catharanthus roseus. In this paper, we identified the amino acid domains necessary for the nuclear targeting of CrMYC2.

Findings: We examined the intracellular localization of whole CrMYC2 and of various deletion mutants, all fused with GFP, using a transient expression assay in onion epidermal cells. Sequence analysis of this protein revealed the presence of four putative basic nuclear localization signals (NLS). Assays showed that none of the predicted NLS is active alone. Further functional dissection of CrMYC2 showed that the nuclear targeting of this transcription factor involves the cooperation of three domains located in the C-terminal region of the protein. The first two domains are located at amino acid residues 454-510 and 510-562 and contain basic classical monopartite NLSs; these regions are referred to as NLS3 (KRPRKR) and NLS4 (EAERQRREK), respectively. The third domain, between residues 617 and 652, is rich in basic amino acids that are well conserved in other phylogenetically related bHLH transcription factors. Our data revealed that these three domains are inactive when isolated but act cooperatively to target CrMYC2 to the nucleus.

Conclusions: This study identified three amino acid domains that act in cooperation to target the CrMYC2 transcription factor to the nucleus. Further fine structure/function analysis of these amino acid domains will allow the identification of new NLS domains and will allow the investigation of the related molecular mechanisms involved in the nuclear targeting of the CrMYC2 bHLH transcription factor.

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Figures

Figure 1
Figure 1

Schematic representation of the full-length CrMYC2 protein showing the positions of the potential nuclear localization signal sequences (NLS 1, 2, 3, and 4) and of the regions of CrMYC2 used to make the GFP fusions. NLS: nuclear localization signal, CrMYC2: full-length CrMYC2 sequence, F1 to F8: partial CrMYC2 sequences used for fusion to GFP. Grey box: bHLH domain. Single oval shape: monopartite NLS. Double oval shape: bipartite NLS.

Figure 2
Figure 2

Alignment of amino acid sequences of CrMYC2, AtMYC2/RAP1/AtbHLH006/At1g32640.1, At4g17880.1, At5g46760.1, At5g46830.1 and Os10g42430.1. Bold letters correspond to conserved amino acids residues within a putative NLS. Basic amino acids are boxed in grey. Asterisks under sequences correspond to positions where the amino acid is the same for all sequences. Boxed or underlined sequences indicate the three regions involved in CrMYC2 nuclear targeting.

Figure 3
Figure 3

Subcellular localization in epidermal onion cells of fusions of GFP with full-length CrMYC2, with different putative nuclear localization signals and with deletion mutants of CrMYC2. The green fluorescence of the GFP protein was visualized by epifluorescence microscopy. A: control with GFP alone. B: control with GFP fused to the NLS of the SV40 large T antigen. C: control with GFP fused to an endoplasmic reticulum retention signal. D: right, GFP fused to full-length CrMYC2, left: corresponding DAPI staining,. E-H: GFP fused with different putative NLS sequences from CrMYC2, 1, 2, 3 and 4, respectively; I-P: GFP fused with different deletion mutants of CrMYC2 (F1 to F8).

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