The AAA-ATPase p97 in mitosis and fertilization

Late mitotic events are chiefly controlled by proteolysis of key
regulatory proteins via the ubiquitin-proteasome pathway. In this
pathway ubiquitin ligases modify substrates by attachment of
ubiquitin (“ubiquitylation”), which usually results in their
subsequent degradation by the 26S proteasome. The crucial ubiquitin
ligase involved in late mitosis is the anaphase-promoting complex
or cyclosome (APC/C). Among the many substrates of the APC/C is the
anaphase inhibitor securin, whose destruction leads to activation
of separase, which in turn triggers sister chromatid separation by
proteolytic cleavage of cohesin. The APC/C also targets cyclin B1,
an activating subunit of Cdk1 kinase, whose inactivation is a
prerequisite for mitotic exit. The unstable APC/C substrates are
often found in association with stable partner proteins. How single
subunits of multi-protein complexes are selectively extracted and
eventually degraded is largely unknown, but there is increasing
evidence that additional factors assist to extract
ubiquitin-carrying subunits from stable binding partners. One such
factor is vertebrate p97 (Cdc48 in yeast), an abundant and highly
conserved member of the AAA-ATPase family. It is involved in such
diverse processes as transcriptional regulation, membrane fusion,
and ER-associated protein degradation (ERAD). The unifying scheme
in these seemingly unrelated functions is that p97 is able to
“extract” preferentially ubiquitylated proteins from their
environment. Roles of p97 in mitosis have recently emerged: p97 was
reported to be required for spindle disassembly and for nuclear
envelope reformation during mitotic exit in Xenopus. Furthermore, a
genetic interaction between p97, separase and securin, as well as a
requirement of p97 for separase stability, were discovered in
fission yeast. Given these hints and the importance of
ubiquitylation in both mitosis and p97 pathways, this study
intended to elucidate additional mitotic roles of p97 in
vertebrates. Towards this end, tools to interfere with p97 function
in Xenopus egg extracts were developed. These included
immunodepletion of the p97 adaptors Npl4, Ufd1 and p47 and addition
of recombinant dominant-negative p97-mutants. ERAD, which could be
established here for the first time in Xenopus egg extracts, was
greatly impaired in the absence of p97 function. However, many
aspects of mitosis were found to be unaffected. Importantly, p97’s
proposed role in spindle disassembly was clearly falsified within
this thesis. Furthermore, p97 was shown to be dispensable for
activity and stability of vertebrate separase. Disassembly of the
mitotic checkpoint complex, which prevents premature APC/C
activation by sequestering its activator Cdc20, did also not
require functional p97 despite its dependence on ubiquitylation of
Cdc20. However, a novel function of p97 at fertilization was
discovered. p97 was found to interact with nucleoplasmin, a
histone-binding chaperone that catalyzes the exchange of
sperm-specific basic proteins (SBPs) to histones. Indeed,
interference with p97 function delayed sperm decondensation in
Xenopus egg extracts, thereby confirming a novel role of this
AAA-ATPase in sperm chromatin remodelling. In another project the
role of securin in human cells was investigated. Human cells
lacking securin had been reported to suffer from massive chromosome
missegregation, which was in sharp contrast to the mild phenotype
of securin knockout mice. In collaboration with the group of M.
Speicher it could be demonstrated that chromosome losses in
securin-/- cells are transient and give way to a stable segregation
pattern after just a few passages. This was despite persisting
biochemical defects such as reduced level and activity of separase.
These data demonstrate that securin is dispensable for chromosomal
stability in human cells.