Nakase, Y. | Search bioRxiv

bioRxiv2023

Cdc48 and its co-factor Ufd1 segregate CENP-A from centromeric chromatin and can induce chromosome elimination in the fission yeast Schizosaccharomyces pombe.

CENP-A, a variant of histone H3, determines the identity of the centromere, a chromosome locus at which a microtubule attachment site termed kinetochore is assembled. Because the position and size of the centromere and its number per chromosome must be maintained for the faithful segregation of chromosomes, the distribution of CENP-A is strictly regulated. In this study, we have aimed to understand mechanisms to regulate the distribution of CENP-A by a genetic approach in the fission yeast Schizosaccharomyces pombe. A mutant of the ufd1+ gene (ufd1-73) encoding a cofactor of Cdc48 ATPase is sensitive to CENP-A expressed at a high level and allows mislocalization of CENP-A. ChIP analysis has revealed that the level of CENP-A in centromeric chromatin is increased in the ufd1-73 mutant even when CENP-A is expressed at a normal level. A preexisting mutant of the cdc48+ gene (cdc48-353) phenocopies the ufd1-73 mutant. We have also shown that Cdc48 and Ufd1 proteins physically interact with centromeric chromatin. Finally, Cdc48 ATPase with Ufd1 artificially recruited to the centromere of a mini-chromosome (Ch16) induce a loss of CENP-A from Ch16, resulting in an increased rate of chromosome loss. It appears that Cdc48 ATPase, together with its cofactor Ufd1 segregates excess CENP-A from chromatin, likely in a direct manner, to maintain proper distribution of CENP-A. This mechanism may play an important role in centromere disassembly, a process to eliminate CENP-A massively to inactivate the kinetochore function during development, differentiation, and stress response in other organisms. Significance statementMaintaining the proper distribution of CENP-A is crucial for centromere identity. This process involves accurate positioning of CENP-A and removal of excess CENP-A from chromatin. The Cdc48-Ufd1 complex is essential for this regulation as it acts as a segregase that directly eliminates surplus CENP-A from chromatin. These findings have therapeutic significance as targeting the Cdc48 complex can potentially correct abnormal karyotypes in disomic embryos and prevent trisomic disorders like Down syndrome. This research advances our understanding of centromeric chromatin regulation and its potential therapeutic applications.

molecular biology↗

bioRxiv2023

Rapamycin-sensitive mechanisms confine the growth of fission yeast below the temperatures detrimental to cell physiology

Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to protein denaturing and heat damage to other cellular macromolecules. We here report that, in the presence of the macrolide compound rapamycin, the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that normally arrest its growth. Rapamycin is a potent inhibitor of the protein kinase complex TOR Complex 1 (TORC1), and consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance. These results suggest that TORC1, a well-established growth promoter, restricts the high-temperature growth of fission yeast and that compromised TORC1 signaling allows cell proliferation at higher temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens in S. pombe, which identified Sck1 and additional factors that appear to suppress cell proliferation at high temperatures. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology. Thus, growth arrest at high temperatures may not directly result from heat damage to cellular components essential for proliferation and viability. Significance StatementThe immunosuppressant rapamycin is a specific inhibitor of the protein kinase Target Of Rapamycin (TOR), and the drug is known to extend the lifespan of diverse eukaryotic organisms. In this study, we have found that rapamycin confers heat resistance on fission yeast, allowing its proliferation above the normal permissive temperatures. This unexpected observation suggests that TOR, which is known as a growth-promoting kinase, is inhibitory to cell proliferation at high temperatures. Our genome-wide screens have identified additional genes whose deletion leads to improved growth under heat stress. Thus, cells may have mechanisms that restrict proliferation even below the temperatures deleterious to their physiology.

cell biology↗