N -Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

• Published in: International journal of molecular sciences 2020-08, Vol.21 (17), p.6175
• Main Authors: Tan, Hwei Ling, Zeng, Yi Bing, Chen, Ee Sin
• Format: Article
• Language: English
• Subjects: Binding Sites; Cell Cycle Proteins - genetics; Centromeres; Chromatin; Chromosomal Proteins, Non-Histone - chemistry; Chromosomal Proteins, Non-Histone - genetics; Chromosomal Proteins, Non-Histone - metabolism; Chromosomes; Deoxyribonucleic acid; DNA; Domains; Epigenetics; Experiments; Genomes; Heredity; Heterochromatin; Heterochromatin - metabolism; Histone H3; Histone-Lysine N-Methyltransferase - genetics; Histones; Lysine; Methyltransferase; Microtubules; Mutants; Mutation; N-Terminus; Nuclear Proteins - metabolism; Nucleotide sequence; Proline; Protein A; Protein Binding; Protein Domains; Protein Stability; Protein turnover; Proteins; Proteolysis; Schizosaccharomyces - chemistry; Schizosaccharomyces - genetics; Schizosaccharomyces - metabolism; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - chemistry; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Stability; Yeast
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21176175

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 ( )-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 CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of 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 ( ), 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.