Live-cell visualization of intracellular interaction between a nuclear migration protein (hNUDC) and the
Yuan-Bin Zheng1, Ying-Ying Xiao, Peng Tan
1The Key Laboratory of Gene Engineering of Education Ministry, Zhongshan University, Guangzhou, The People's Republic of China.
Abstract:
We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.
Insights
The human NUDC protein interacts with Mpl, forming a complex that directs NUDC to the cell surface for secretion. This interaction, mediated by NUDC's N-terminal region, is crucial for NUDC secretion.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Interactions
Background:
- Endogenous human NUDC (hNUDC) and Mpl were previously observed to co-localize in megakaryocyte cells.
- hNUDC alone accumulates in the Golgi, but co-expression with Mpl shifts its localization to the cytosol.
Purpose of the Study:
- To investigate if a complex between hNUDC and Mpl alters hNUDC's subcellular location and induces secretion.
- To identify the specific region of hNUDC responsible for Mpl binding and subsequent secretion.
Main Methods:
- Bimolecular fluorescence complementation (BiFC) was used to visualize hNUDC/Mpl complex formation in living cells.
- Co-transfection with subcellular markers examined the complex's distribution in the endoplasmic reticulum, Golgi, and cell surface.
- Adenovirus-mediated overexpression and siRNA were employed in Dami cells to assess NUDC secretion and Mpl's role.
Main Results:
- The N-terminal 159 amino acids of hNUDC, not the C-terminal half, were found to bind Mpl in vivo.
- Overexpression of hNUDC or its N-terminal fragment in Dami cells increased hNUDC secretion.
- Depletion of Mpl significantly inhibited hNUDC secretion, indicating Mpl's essential role.
Conclusions:
- The N-terminal region of hNUDC contains the necessary information to complex with Mpl.
- The hNUDC/Mpl complex is trafficked through the secretory pathway, leading to hNUDC secretion.
- Mpl binding is critical for the secretion of hNUDC.
