<i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A
Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on whi...
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MDPI AG
2020-08-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/21/17/6175 |
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| author | Hwei Ling Tan Yi Bing Zeng Ee Sin Chen |
| author_facet | Hwei Ling Tan Yi Bing Zeng Ee Sin Chen |
| author_sort | Hwei Ling Tan |
| collection | DOAJ |
| description | Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (<i>N</i>)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in <i>Saccharomyces cerevisiae</i> CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of <i>Schizosaccharomyces pombe</i> CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (<i>Δclr4</i>), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover. |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T16:47:05Z |
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| spelling | doaj.art-9dc129af99904cc8b5d6892c4003b5b42023-11-20T11:30:16ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672020-08-012117617510.3390/ijms21176175<i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-AHwei Ling Tan0Yi Bing Zeng1Ee Sin Chen2Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, SingaporeDepartment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, SingaporeDepartment of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117596, SingaporeCentromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (<i>N</i>)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in <i>Saccharomyces cerevisiae</i> CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of <i>Schizosaccharomyces pombe</i> CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (<i>Δclr4</i>), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.https://www.mdpi.com/1422-0067/21/17/6175CENP-Acentromerechromosome segregationfission yeast<i>N</i>-terminusprotein turnover |
| spellingShingle | Hwei Ling Tan Yi Bing Zeng Ee Sin Chen <i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A International Journal of Molecular Sciences CENP-A centromere chromosome segregation fission yeast <i>N</i>-terminus protein turnover |
| title | <i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A |
| title_full | <i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A |
| title_fullStr | <i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A |
| title_full_unstemmed | <i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A |
| title_short | <i>N</i>-Terminus Does Not Govern Protein Turnover of <i>Schizosaccharomyces pombe</i> CENP-A |
| title_sort | i n i terminus does not govern protein turnover of i schizosaccharomyces pombe i cenp a |
| topic | CENP-A centromere chromosome segregation fission yeast <i>N</i>-terminus protein turnover |
| url | https://www.mdpi.com/1422-0067/21/17/6175 |
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