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Functional Analysis of Hif1 Histone Chaperone in Saccharomyces cerevisiae.

ABSTRACT: The Hif1 protein in the yeast Saccharomyces cerevisie is an evolutionarily conserved H3/H4-specific chaperone and a subunit of the nuclear Hat1 complex that catalyzes the acetylation of newly synthesized histone H4. Hif1, as well as its human homolog NASP, has been implicated in an array of chromatin-related processes including histone H3/H4 transport, chromatin assembly and DNA repair. In this study, we elucidate the functional aspects of Hif1 Initially we establish the wide distribution of Hif1 homologs with an evolutionarily conserved pattern of four tetratricopeptide repeats (TPR) motifs throughout the major fungal lineages and beyond. Subsequently, through targeted mutational analysis, we demonstrate that the acidic region that interrupts the TPR2 is essential for Hif1 physical interactions with the Hat1/Hat2-complex, Asf1, and with histones H3/H4. Furthermore, we provide evidence for the involvement of Hif1 in regulation of histone metabolism by showing that cells lacking HIF1 are both sensitive to histone H3 over expression, as well as synthetic lethal with a deletion of histone mRNA regulator LSM1 We also show that a basic patch present at the extreme C-terminus of Hif1 is essential for its proper nuclear localization. Finally, we describe a physical interaction with a transcriptional regulatory protein Spt2, possibly linking Hif1 and the Hat1 complex to transcription-associated chromatin reassembly. Taken together, our results provide novel mechanistic insights into Hif1 functions and establish it as an important protein in chromatin-associated processes.

SUBMITTER: Dannah NS

PROVIDER: S-EPMC5982827 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundHuman BiP is traditionally regarded as a major endoplasmic reticulum (ER) chaperone performing a number of well-described functions in the ER. In recent years it was well established that this molecule can also be located in other cell organelles and compartments, on the cell surface or be secreted. Also novel functions were assigned to this protein. Importantly, BiP protein appears to be involved in cancer and rheumatoid arthritis progression, autoimmune inflammation and tissue damage, and thus could potentially be used for therapeutic purposes. In addition, a growing body of evidence indicates BiP as a new therapeutic target for the treatment of neurodegenerative diseases. Increasing importance of this protein and its involvement in critical human diseases demands new source of high quality native recombinant human BiP for further studies and potential application. Here we introduce yeast Saccharomyces cerevisiae as a host for the generation of human BiP protein.ResultsExpression of a full-length human BiP precursor in S. cerevisiae resulted in a high-level secretion of mature recombinant protein into the culture medium. The newly discovered ability of the yeast cells to recognize, correctly process the native signal sequence of human BiP and secrete this protein into the growth media allowed simple one-step purification of highly pure recombinant BiP protein with yields reaching 10 mg/L. Data presented in this study shows that secreted recombinant human BiP possesses native amino acid sequence and structural integrity, is biologically active and without yeast-derived modifications. Strikingly, ATPase activity of yeast-derived human BiP protein exceeded the activity of E. coli-derived recombinant human BiP by a 3-fold.ConclusionsS. cerevisiae is able to correctly process and secrete human BiP protein. Consequently, resulting recombinant BiP protein corresponds accurately to native analogue. The ability to produce large quantities of native recombinant human BiP in yeast expression system should accelerate the analysis and application of this important protein.

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Functional Analysis of Hif1 Histone Chaperone in Saccharomyces cerevisiae.

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