Limits...
A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

Jia H, Zhang X, Wang W, Bai Y, Ling Y, Cao C, Ma RZ, Zhong H, Wang X, Xu Q - BMC Cell Biol. (2015)

Bottom Line: Consistent with this speculation, a direct association between Mps1 and Crm1 was found.Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm.Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

View Article: PubMed Central - PubMed

Affiliation: Navy General Hospital of China, Beijing, 100048, China. 13321120266@126.com.

ABSTRACT

Background: Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear.

Results: In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm.

Conclusion: Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

Show MeSH

Related in: MedlinePlus

Mps1 bears a putative nuclear export sequence. (A) Prediction of nuclear export sequence in Mps1 via 2 online procedures. Mps1 protein sequence was loaded into procedures according to the protocol by software developer. The resulted nuclear sequences were showed as indicated. (B, C) Functional validation of the putative NES. The putative sequences were fused with EGFP in frame and the resulted constructs were transfected into 293 T cells. The subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated. DNA was counterstained with PI. The ratio of fusion proteins in the nucleus and cytoplasm were measured via image J and the statistical result of these were generated by using Graphpad software. (D) The amino acids alignment of NES motifs. The pNES1 of Mps1 is aligned with the classical NES from MAPKK, Cyclin B1, HIV-REV. The letters in red indicate identical or similar amino acids. PI, Propidium Iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4373099&req=5

Fig2: Mps1 bears a putative nuclear export sequence. (A) Prediction of nuclear export sequence in Mps1 via 2 online procedures. Mps1 protein sequence was loaded into procedures according to the protocol by software developer. The resulted nuclear sequences were showed as indicated. (B, C) Functional validation of the putative NES. The putative sequences were fused with EGFP in frame and the resulted constructs were transfected into 293 T cells. The subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated. DNA was counterstained with PI. The ratio of fusion proteins in the nucleus and cytoplasm were measured via image J and the statistical result of these were generated by using Graphpad software. (D) The amino acids alignment of NES motifs. The pNES1 of Mps1 is aligned with the classical NES from MAPKK, Cyclin B1, HIV-REV. The letters in red indicate identical or similar amino acids. PI, Propidium Iodide.

Mentions: Crm1 carries its cargo proteins through binding to a nuclear export sequence. Given the direct interaction of Mps1 and Crm1, it was speculated that Mps1 bears the nuclear export sequence(s). To uncover the putative NES, the Mps1 protein sequence was analyzed via 2 online software programs NetNES (www.cbs.dtu.dk/services/NetNES/) and ELM (http://elm.eu.org/links.html). As shown in Figure 2A, only 1 putative NES was revealed in both ELM and NetNES. To validate these prediction sequences, these putative NES sequences were fused to the N-terminal of enhanced-green fluorescence protein (EGFP) and the distribution of the resulted fusion protein was examined after a transient transfection of 293 T cells. Only the pNES1 fused EGFP resided in the cytoplasm while the other 2 pNES fusion proteins showed a similar distribution pattern as that of the EGFP. The cytoplasmic distribution of pNES1-EGFP appears to rely on Crm1, as the treatment of cells with LMB caused the nuclear enrichment of pNES1-fused EGFP (Figure 2B and C). The pNES1 seemed to be a classic NES sequence as the leucines enriched pattern is conserved when aligned to the classic NES sequence of Cyclin B1, HIV Rev and MAPK (Figure 2D). Notably, replacement of these conserved leucines with alanine caused re-localization of EGFP into the nucleus (Additional file 2: Figure S1). Based on these findings, it is thought that pNES1 in Mps1 is a functional NES.Figure 2


A putative N-terminal nuclear export sequence is sufficient for Mps1 nuclear exclusion during interphase.

Jia H, Zhang X, Wang W, Bai Y, Ling Y, Cao C, Ma RZ, Zhong H, Wang X, Xu Q - BMC Cell Biol. (2015)

Mps1 bears a putative nuclear export sequence. (A) Prediction of nuclear export sequence in Mps1 via 2 online procedures. Mps1 protein sequence was loaded into procedures according to the protocol by software developer. The resulted nuclear sequences were showed as indicated. (B, C) Functional validation of the putative NES. The putative sequences were fused with EGFP in frame and the resulted constructs were transfected into 293 T cells. The subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated. DNA was counterstained with PI. The ratio of fusion proteins in the nucleus and cytoplasm were measured via image J and the statistical result of these were generated by using Graphpad software. (D) The amino acids alignment of NES motifs. The pNES1 of Mps1 is aligned with the classical NES from MAPKK, Cyclin B1, HIV-REV. The letters in red indicate identical or similar amino acids. PI, Propidium Iodide.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4373099&req=5

Fig2: Mps1 bears a putative nuclear export sequence. (A) Prediction of nuclear export sequence in Mps1 via 2 online procedures. Mps1 protein sequence was loaded into procedures according to the protocol by software developer. The resulted nuclear sequences were showed as indicated. (B, C) Functional validation of the putative NES. The putative sequences were fused with EGFP in frame and the resulted constructs were transfected into 293 T cells. The subcellular distribution of fusion proteins were examined after 48 h under the conditions indicated. DNA was counterstained with PI. The ratio of fusion proteins in the nucleus and cytoplasm were measured via image J and the statistical result of these were generated by using Graphpad software. (D) The amino acids alignment of NES motifs. The pNES1 of Mps1 is aligned with the classical NES from MAPKK, Cyclin B1, HIV-REV. The letters in red indicate identical or similar amino acids. PI, Propidium Iodide.
Mentions: Crm1 carries its cargo proteins through binding to a nuclear export sequence. Given the direct interaction of Mps1 and Crm1, it was speculated that Mps1 bears the nuclear export sequence(s). To uncover the putative NES, the Mps1 protein sequence was analyzed via 2 online software programs NetNES (www.cbs.dtu.dk/services/NetNES/) and ELM (http://elm.eu.org/links.html). As shown in Figure 2A, only 1 putative NES was revealed in both ELM and NetNES. To validate these prediction sequences, these putative NES sequences were fused to the N-terminal of enhanced-green fluorescence protein (EGFP) and the distribution of the resulted fusion protein was examined after a transient transfection of 293 T cells. Only the pNES1 fused EGFP resided in the cytoplasm while the other 2 pNES fusion proteins showed a similar distribution pattern as that of the EGFP. The cytoplasmic distribution of pNES1-EGFP appears to rely on Crm1, as the treatment of cells with LMB caused the nuclear enrichment of pNES1-fused EGFP (Figure 2B and C). The pNES1 seemed to be a classic NES sequence as the leucines enriched pattern is conserved when aligned to the classic NES sequence of Cyclin B1, HIV Rev and MAPK (Figure 2D). Notably, replacement of these conserved leucines with alanine caused re-localization of EGFP into the nucleus (Additional file 2: Figure S1). Based on these findings, it is thought that pNES1 in Mps1 is a functional NES.Figure 2

Bottom Line: Consistent with this speculation, a direct association between Mps1 and Crm1 was found.Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm.Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

View Article: PubMed Central - PubMed

Affiliation: Navy General Hospital of China, Beijing, 100048, China. 13321120266@126.com.

ABSTRACT

Background: Mps1, an essential component of the mitotic checkpoint, is also an important interphase regulator and has roles in DNA damage response, cytokinesis and centrosome duplication. Mps1 predominantly resides in the cytoplasm and relocates into the nucleus at the late G2 phase. So far, the mechanism underlying the Mps1 translocation between the cytoplasm and nucleus has been unclear.

Results: In this work, a dynamic export process of Mps1 from the nucleus to cytoplasm in interphase was revealed- a process blocked by the Crm1 inhibitor, Leptomycin B, suggesting that export of Mps1 is Crm1 dependent. Consistent with this speculation, a direct association between Mps1 and Crm1 was found. Furthermore, a putative nuclear export sequence (pNES) motif at the N-terminal of Mps1 was identified by analyzing the motif of Mps1. This motif shows a high sequence similarity to the classic NES, a fusion of this motif with EGFP results in dramatic exclusion of the fusion protein from the nucleus. Additionally, Mps1 mutant loss of pNES integrity was shown by replacing leucine with alanine which produced a diffused subcellular distribution, compared to the wild type protein which resides predominantly in cytoplasm.

Conclusion: Taken these findings together, it was concluded that the pNES sequence is sufficient for the Mps1 export from nucleus during interphase.

Show MeSH
Related in: MedlinePlus