Assembly and sorting mechanisms of CCAAT-box binding complex of filamentous fungi

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One of the transcription factors of filamentous fungi, CCAAT-box binding factor, is widely conserved among eukaryotes, which consist of three subunits HapB, and C and E. The following results were obtained in this research.1. The order of the assembly of CCAAT-box binding complex subunits was determined.2. All three Hap subunits were expressed as a GFP-fusion protein in the hap deletion strains, respectively. Observation of the localization of the GFP-fusion proteins revealed that nuclear localization of HapC and HapE requires the other two subunits, respectively, while nuclear translocation of HapB was independent on HapC and HapE.3. Analysis on the nuclear localization signal of HapB revealed that there are two redundant nuclear localization signals in the conserved core region and non-conserved C-terminal region.4. It was found that HapE is easy to become an insoluble form by itself. Addition of HapC prior to the in vitro synthesis of HapE significantly increases the solubility of HapE. Taken together with the facts that HapE and HapC possess the histone-fold motif and form a hetero-dimer, it was suggested that HapC may act as a chaperon-like factor specific to HapE to prevent the insolublilization of HapE.In this research, we have elucidated the mechanism of the assembly-dependent nuclear localization of the filamentous fungal CCAAT-box binding factor which universally exists in eukaryotes, prior to researches with other eukaryotic organisms. Furthermore, we have also elucidated the molecule mechanism of the stabilization of the HapE by HapC, which is a potential molecular basis for the quantity control mechanisms of the subunits of the CCAAT-binding complex.