PubMed · 4765872
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1973-11-10. Editorial: Hela cells.. https://doi.org/10.5694/j.1326-5377.1973.tb76610.x
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Components of multiprotein complexes are routinely determined by using proteomic approaches. However, this information lacks functional content except when new complex members are identified. To analyze quantitatively the abundance of proteins in human Mediator we used normalized spectral abundance factors generated from shotgun proteomics data sets. With this approach we define a common core of mammalian Mediator subunits shared by alternative forms that variably associate with the kinase module and RNA polymerase (pol) II. Although each version of affinity-purified Mediator contained some kinase module and RNA pol II, Mediator purified through F-Med26 contained the most RNA pol II and the least kinase module as demonstrated by the normalized spectral abundance factor approach. The distinct forms of Mediator were functionally characterized by using a transcriptional activity assay, where F-Med26 Mediator/RNA pol II was the most active. This method of protein complex visualization has important implications for the analysis of multiprotein complexes and assembly of protein interaction networks.
The ubiquitin system plays important roles in the regulation of numerous cellular processes by conjugating ubiquitin to target proteins. In most cases, conjugation of polyubiquitin to target proteins regulates their function. In the polyubiquitin chains reported to date, ubiquitin monomers are linked via isopeptide bonds between an internal Lys and a C-terminal Gly. Here, we report that a protein complex consisting of two RING finger proteins, HOIL-1L and HOIP, exhibits ubiquitin polymerization activity by recognizing ubiquitin moieties of proteins. The polyubiquitin chain generated by the complex is not formed by Lys linkages, but by linkages between the C- and N-termini of ubiquitin, indicating that the ligase complex possesses a unique feature to assemble a novel head-to-tail linear polyubiquitin chain. Moreover, the complex regulates the stability of Ub-GFP (a GFP fusion protein with an N-terminal ubiquitin). The linear polyubiquitin chain generated post-translationally may function as a new modulator of proteins.
To elucidate whether phosphorylation of myosin II regulatory light chain (MRLC) is essential for myosin II recruitment to the furrow during cytokinesis, HeLa cells transfected with three types of GFP-tagged recombinant MRLCs, wild-type MRLC, non-phosphorylated form of MRLC, and phosphorylated form of MRLC, were examined. Living cell-imaging showed that both phosphorylated and non-phosphorylated form of MRLCs were recruited to the equator at the same time after anaphase onset, suggesting that phosphorylation of MRLC is not responsible for recruitment of myosin II to the equator. Moreover, the treatment with an inhibitor of myosin II activity, blebbistatin, induced no effect on recruitment of those three recombinant MRLCs. During cytokinesis, phosphorylated but not non-phosphorylated form of MRLC was retained in the equator. These results suggest that phosphorylation of MRLC is essential for retainment of myosin II in the furrow but not for initial recruitment of myosin II to the furrow in dividing HeLa cells.