The nuclear matrix has classically been assumed to be a solid structure coherently aligning nuclear components, but its real nature remains obscure. We separated the proteins in a ribonucleoproteincontaining nuclear matrix fraction of HeLa cells by reversed-phase HPLC followed by SDS-PAGE, and identified 83 proteins through peptide mass fingerprint (PMF) analysis. Many nucleolar proteins, classical nuclear matrix proteins, RNA binding proteins, cytoskeletal proteins and five uncharacterized proteins were identified in this fraction. Four of the latter proteins were localized to the cell nucleus, BXDC1 and EBNA1BP2 being especially localized to the nucleolus. Fluorescence recovery after photobleaching and RNAi knockdown analyses suggested that BXDC1 and EBNA1BP2 function in a dynamic scaffold for ribosome biogenesis.
To find novel proteins predicted to participate in the formation of nuclear bodies, nuclear speckles, and nuclear macro-protein complexes, we applied proteome analysis to a HeLa cell nuclear matrix fraction. Proteins in the fraction were separated by SDS-PAGE, digested with trypsin, and analyzed by nanoflow liquid chromatography-iontrap-tandem mass spectrometry. Three hundred and thirty three proteins including 39 novel ones were identified. Seven WD-repeat proteins and 16 disordered region-rich proteins, which act frequently as scaffolding proteins for macro-protein complexes, were found amongst the novel proteins.
The LEM (LAP2beta, Emerin, and MAN1) proteins are essential for nuclear membrane targeting to chromatin via an association with barrier-to-autointegration factor (BAF). Herein, we focused on the mitotic phosphorylation of MAN1 and its biological role. MAN1 was phosphorylated in a cell cycle-dependent manner in the Xenopus egg cell-free system, and the mitotic phosphorylation at the N-terminal region of MAN1 suppressed the binding of MAN1 to BAF. Titansphere column chromatography followed by MS/MS sequencing identified at least three M-phase-specific phosphorylation sites, Thr-209, Ser-351, and Ser-402, and one cell cycle-independent phosphorylation site, Ser-463. An in vitro BAF binding assay involving mutants S402A and S402E suggested that the phosphorylation of Ser-402 was important for regulation of the binding of MAN1 to BAF.
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