RESUMO
Cse4p is an evolutionarily conserved histone H3-like protein that is thought to replace H3 in a specialized nucleosome at the yeast (Saccharomyces cerevisiae) centromere. All known yeast, worm, fly, and human centromere H3-like proteins have highly conserved C-terminal histone fold domains (HFD) but very different N termini. We have carried out a comprehensive and systematic mutagenesis of the Cse4p N terminus to analyze its function. Surprisingly, only a 33-amino-acid domain within the 130-amino-acid-long N terminus is required for Cse4p N-terminal function. The spacing of the essential N-terminal domain (END) relative to the HFD can be changed significantly without an apparent effect on Cse4p function. The END appears to be important for interactions between Cse4p and known kinetochore components, including the Ctf19p/Mcm21p/Okp1p complex. Genetic and biochemical evidence shows that Cse4p proteins interact with each other in vivo and that nonfunctional cse4 END and HFD mutant proteins can form functional mixed complexes. These results support different roles for the Cse4p N terminus and the HFD in centromere function and are consistent with the proposed Cse4p nucleosome model. The structure-function characteristics of the Cse4p N terminus are relevant to understanding how other H3-like proteins, such as the human homolog CENP-A, function in kinetochore assembly and chromosome segregation.
Assuntos
Proteínas de Ciclo Celular , Centrômero/metabolismo , Cromatina/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Histonas/fisiologia , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Alanina , Alelos , Sequência de Aminoácidos , Sítios de Ligação , Cromatina/genética , Proteínas Cromossômicas não Histona , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Histonas/genética , Cinetocoros , Dados de Sequência Molecular , Mutagênese , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A.
Assuntos
Autoantígenos , Centrômero/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Sequência de Bases , Proteína Centromérica A , Cromatina/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Expressão Gênica , Genes Fúngicos , Variação Genética , Histonas/genética , Humanos , Mitose , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Dobramento de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
A chlamydial glycolipid antigen (GLXA) is shed into the medium of C. trachomatis-infected cell cultures. This study screened monoclonal antibodies (mAb), prepared in different laboratories by immunization with embryonated egg propagated elementary bodies (EB), for their ability to bind with infected cells and to react with purified GLXA isolated from supernatants of infected McCoy cells. The fluorescent antibody (FA) staining pattern exhibited by a number of mAb indicated that they bound antigen present within the inclusion and at the inner membrane surface of infected cells; the observed pattern differs significantly from the distribution seen when anti-lipopolysaccharide (LPS) (mAb) were used. The staining pattern observed by immunofluorescence was confirmed and extended by ultrastructure studies of immunogold-labelled, infected human endometrial gland epithelial cells (HEGEC) and a human endometrial carcinoma-derived cell line (RL95-2). Additionally, the immunoelectron microscope studies revealed binding within the inclusion and on reticulate bodies, within the cell cytoplasm and at the surface of infected cells. The specificity of the reactive mAb, examined by molecular shift chromatography and isolated, affinity-purified GLXA, indicated that two mAb of the IgG isotype recognized an antigen which had been purified from tissue culture supernatants by affinity chromatography using an IgM mAb. The results suggest that GLXA is an important determinant whose role and function during in vitro and in vivo infections deserves further analyses.
Assuntos
Antígenos de Bactérias/análise , Chlamydia/imunologia , Glicolipídeos/imunologia , Polissacarídeos Bacterianos/imunologia , Anticorpos Monoclonais , Especificidade de Anticorpos/imunologia , Células Cultivadas , Epitopos/análise , Imunofluorescência , Humanos , Imunoglobulina G/imunologia , Microscopia EletrônicaRESUMO
The centromere, a differentiated region of the eukaryotic chromosome, mediates the segregation of sister chromatids at mitosis. In this study, a Saccharomyces cerevisiae chromosome mis-segregation mutant, cse4-1, has been isolated and shown to increase the nondisjunction frequency of a chromosome bearing a mutant centromere DNA sequence. In addition, at elevated temperatures the cse4-1 allele causes a mitosis-specific arrest with a predominance of large budded cells containing single G2 nuclei and short bipolar mitotic spindles. The wild-type gene, CSE4, is essential for cell division and encodes a protein containing a domain that is 64% identical to the highly conserved chromatin protein, histone H3. Biochemical experiments demonstrate that CSE4p has similar DNA-binding characteristics as those of histone H3 and might form a specialized nucleosome structure in vivo. Interestingly, the human centromere protein, CENP-A, also contains this H3-like domain. Data presented here indicate that CSE4p is required for proper kinetochore function in yeast and may represent an evolutionarily conserved protein necessary for assembly of the unique chromatin structure associated with the eukaryotic centromere.