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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.

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Related in: MedlinePlus

Mps1 with all leucines substituted in pNES1 reside in the cytoplasm. (A) Schematic of Mps1 mutant with mutated pNES1 (Mps1mutNES1). The lysines in red were mutated to alanines (upper panel). The sequencing result of the mutated pNES1 (middle and lower panel). (B, C) The subcellular distribution of YFP fused Mps1 and Mps1mutNES1 in presence of LMB and DMSO. The quantitative result of fusion distribution was generated as in Figure 2C. (D) Western blot of 293 T cells shows the expression of YFPMps1 and YFPMps1mutNES1 in SW480 cell lines.
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Fig3: Mps1 with all leucines substituted in pNES1 reside in the cytoplasm. (A) Schematic of Mps1 mutant with mutated pNES1 (Mps1mutNES1). The lysines in red were mutated to alanines (upper panel). The sequencing result of the mutated pNES1 (middle and lower panel). (B, C) The subcellular distribution of YFP fused Mps1 and Mps1mutNES1 in presence of LMB and DMSO. The quantitative result of fusion distribution was generated as in Figure 2C. (D) Western blot of 293 T cells shows the expression of YFPMps1 and YFPMps1mutNES1 in SW480 cell lines.

Mentions: To further validate that pNES1 is a functional NES, The subcellular distribution of Mps1 mutants, in which 1 isoleucine and 3 leucines were replaced with alanines (Mps1mutNES1) was determined. The resulted fusion protein YFPMps1mutNES1 was forcibly expressed in 293 T cells and the subcellular distribution was examined under the indications as shown (Figure 3A, D). Unexpectedly, the Mps1mutNES1 did not reside in the nucleus, even in the presence of LMB (Figure 3B and C), suggesting that pNES1 may be not sufficient for nuclear export, or that this mutation may affect both the nuclear import and export of Mps1 in interphase.Figure 3


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 with all leucines substituted in pNES1 reside in the cytoplasm. (A) Schematic of Mps1 mutant with mutated pNES1 (Mps1mutNES1). The lysines in red were mutated to alanines (upper panel). The sequencing result of the mutated pNES1 (middle and lower panel). (B, C) The subcellular distribution of YFP fused Mps1 and Mps1mutNES1 in presence of LMB and DMSO. The quantitative result of fusion distribution was generated as in Figure 2C. (D) Western blot of 293 T cells shows the expression of YFPMps1 and YFPMps1mutNES1 in SW480 cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Mps1 with all leucines substituted in pNES1 reside in the cytoplasm. (A) Schematic of Mps1 mutant with mutated pNES1 (Mps1mutNES1). The lysines in red were mutated to alanines (upper panel). The sequencing result of the mutated pNES1 (middle and lower panel). (B, C) The subcellular distribution of YFP fused Mps1 and Mps1mutNES1 in presence of LMB and DMSO. The quantitative result of fusion distribution was generated as in Figure 2C. (D) Western blot of 293 T cells shows the expression of YFPMps1 and YFPMps1mutNES1 in SW480 cell lines.
Mentions: To further validate that pNES1 is a functional NES, The subcellular distribution of Mps1 mutants, in which 1 isoleucine and 3 leucines were replaced with alanines (Mps1mutNES1) was determined. The resulted fusion protein YFPMps1mutNES1 was forcibly expressed in 293 T cells and the subcellular distribution was examined under the indications as shown (Figure 3A, D). Unexpectedly, the Mps1mutNES1 did not reside in the nucleus, even in the presence of LMB (Figure 3B and C), suggesting that pNES1 may be not sufficient for nuclear export, or that this mutation may affect both the nuclear import and export of Mps1 in interphase.Figure 3

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