Cell cycle progression requires the CDC-48 ^{UFD−1/NPL−4} complex for efficient DNA replication

Abstract

Since cdc48 mutants were isolated by the first genetic screens for cell division cycle (cdc) mutants in yeast, the requirement of the chaperone-like ATPase Cdc48/p97 during cell division has remained unclear. Here, we discover an unanticipated function for Caenorhabditis elegans CDC-48 in DNA replication linked to cell cycle control. Our analysis of the CDC-48 UFD؊1/NPL؊4 complex identified a general role in S phase progression of mitotic cells essential for embryonic cell division and germline development of adult worms. These developmental defects result from activation of the DNA replication checkpoint caused by replication stress. Similar to loss of replication licensing factors, DNA content is strongly reduced in worms depleted for CDC-48, UFD-1, and NPL-4. In addition, these worms show decreased DNA synthesis and hypersensitivity toward replication blocking agents. Our findings identified a role for CDC-48 UFD؊1/NPL؊4 in DNA replication, which is important for cell cycle progression and genome stability.ATL-1/ATR ͉ C. elegans ͉ CDC-48/p97 ͉ genome stability M any biological processes including development and cell division are tightly controlled by ubiquitin-mediated protein degradation. A central factor for mobilizing and targeting ubiquitylated substrates to the 26S proteasome is Cdc48/p97 (Cdc48 in yeast, CDC-48 in C. elegans, p97 in mammals), a chaperone-like AAA ATPase (1). Its activity is modulated by alternative adaptor proteins, which determine recruitment and processing of specific substrates. Cdc48/p97 forms a complex with the cofactors Ufd1 and Npl4 that is involved in endoplasmic reticulum (ER)-associated protein degradation (ERAD) (2), membrane fusion and cell cycle progression (3, 4). Temperature sensitive cdc48 mutants have already been isolated by early cdc-screens (5) in Saccharomyces cerevisiae. However, the essential role of Cdc48 during cell cycle progression remained elusive.Meanwhile, different activities of Cdc48/p97 in mitosis have been addressed by several studies. Early observations in yeast and recent findings using Xenopus egg extracts suggested that Cdc48/p97 regulates spindle disassembly during exit from mitosis (6, 7). For example, spindle regulators such as the Polo-like kinase Plx remain attached and probably stabilize the spindle in the absence of p97 Ufd1/Npl4 . However, contradictory evidence exists concerning a specific role of the p97 Ufd1/Npl4 complex in spindle dynamics (8,9). Beside spindle function, p97 together with its Ufd1-Npl4 cofactor is important for nuclear envelope assembly (10). Interestingly, it has been shown that p97 stimulates nucleus reformation after mitosis by extracting and thereby inactivating the mitotic progression kinase Aurora B from chromatin (11). Together, these diverse processes involving Cdc48/p97 suggest the existence of multiple substrates that need to be regulated during mitosis.Recently, we found that the C. elegans Cdc48/p97 homologues CDC-48.1 and CDC-48.2 form an evolutionarily conserved complex with UFD-1 and NPL-4 important for t...

Cdc48/p97–Ufd1–Npl4 antagonizes Aurora B during chromosome segregation in HeLa cells

“…A role for Cdc48/p97-Ufd1-Npl4 in mitosis was recently questioned, as a mitotic delay could not be observed in C. elegans embryos treated with RNAi against Cdc48/p97 or Ufd1-Npl4 (Mouysset et al, 2008). However, the RNAi in that study was fed to the animals rather than injected, which can limit efficacy.…”

“…This interpretation for a positive regulation of Aurora B is in surprising contrast with our observation in Xenopus egg extracts (Ramadan et al, 2007), where Cdc48/p97 removes Aurora B from chromosomes. Two other studies using RNAi-mediated depletion of Cdc48/p97 in Caenorhabditis elegans failed to detect any role for Cdc48/p97 or Ufd1-Npl4 in mitosis (Heallen et al, 2008;Mouysset et al, 2008). The cellular relevance of the Cdc48/p97-Ufd1-Npl4 complex for regulation of mitosis and its functional relation to Aurora B has therefore remained controversial.…”

The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development

“…For example, DNA segregation is often impaired when DNA replication is compromised (Mouysset et al, 2008) and this phenotype is exacerbated in the absence of a functional DNA replication checkpoint (Brauchle et al, 2003). Massive DNA segregation defects were not apparent in lrr-1(tm3543) or atl-1(RNAi); lrr-1(tm3543) embryos, although some minor DNA bridges were detected by spinning-disk microscopy in the latter case (data not shown).…”

The p97 ATPase associates with EEA1 to regulate the size of early endosomes

“…It is thought to function as an ubiquitin-selective chaperone to segregate substrate proteins from large immobile cellular complexes, such as the ER membrane, chromatin and microtubule. Accordingly, p97/Cdc48 has been demonstrated to act in variable cellular settings including endoplasmic reticulumassociated protein degradation (ERAD), DNA replication, transcription regulation, the formation of nuclear envelop, spindle disassembly, and the homotypic fusion of ER/Golgi membranes [3][4][5][6][7]. The functional flexibility of p97/Cdc48 is hinged on the various cofactors that bind p97 via its N-terminal domain.…”