Abstract
Lamin B receptor (LBR), a chromatin and lamin B-binding protein in the inner nuclear membrane, has been proposed to target the membrane precursor vesicles to chromatin mediated by importin β during the nuclear envelope (NE) assembly. However, the mechanisms for the binding of LBR with importin β and the membrane targeting by LBR in NE assembly remain largely unknown. In this report, we show that the amino acids (aa) 69-90 of LBR sequences are required to bind with importin β at aa 45-462, and the binding is essential for the NE membrane precursor vesicle targeting to the chromatin during the NE assembly at the end of mitosis. We also show that this binding is cell cycle-regulated and dependent on the phosphorylation of LBR Ser-71 by p34(cdc2) kinase. RNAi knockdown of LBR causes the NE assembly failure and abnormal chromatin decondensation of the daughter cell nuclei, leading to the daughter cell death at early G(1) phase by apoptosis. Perturbation of the interaction of LBR with importin β by deleting the LBR N-terminal spanning region or aa 69-73 also induces the NE assembly failure, the abnormal chromatin decondensation, and the daughter cell death. The first transmembrane domain of LBR promotes the NE production and expansion, because overexpressing this domain is sufficient to induce membrane overproduction of the NE. Thus, these results demonstrate that LBR targets the membrane precursor vesicles to chromatin by interacting with importin β in a LBR phosphorylation-dependent manner during the NE assembly at the end of mitosis and that the first transmembrane domain of LBR promotes the LBR-bearing membrane production and the NE expansion in interphase.
Highlights
We have recently reported that importin  plays a crucial role in the recruitment of the lamin B receptor (LBR)-bearing nuclear envelope (NE) precursor membrane vesicles to the chromatin surface and the NE assembly through a direct interaction with the N-terminal spanning domain of LBR at amino acids 45–90 [15]
Our results demonstrate that LBR performs a crucial role in recruiting the LBRbearing NE precursor membrane vesicles to the chromatin during the NE assembly mediated by importin  in a phosphorylation-dependent manner in mitosis and that the first transmembrane domain of LBR is responsible for the membrane production and the targeting of this protein to the NE during the nuclear growth and the NE expansion in interphase
The results showed that the endogenous importin  co-precipitated with both green fluorescent protein (GFP)-xLBR1–210 and GFP-hLBR1–210 from the mitotic but not the interphase cell lysates (Fig. 1B)
Summary
Our results demonstrate that LBR performs a crucial role in recruiting the LBRbearing NE precursor membrane vesicles to the chromatin during the NE assembly mediated by importin  in a phosphorylation-dependent manner in mitosis and that the first transmembrane domain of LBR is responsible for the membrane production and the targeting of this protein to the NE during the nuclear growth and the NE expansion in interphase. To investigate how the interaction of LBR with importin  is regulated, we cloned the N-terminal spanning region aa 1–210 of Xenopus and human LBR genes and transiently expressed both GFP-xLBR1–210 and GFPhLBR1–210 in human HeLa cells (Fig. 1).
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