(Received for publication, May 5, 1995; and in revised form, June 6, 1995)
From the
Assembly and disassembly of the nucleus at mitosis in eukaryotes involves the reversible interaction of chromatin with the nuclear membrane. Previously we have shown that this interaction is regulated by the antagonistic activities of a kinase and a phosphatase. The kinase promotes membrane release while the phosphatase stimulates binding. In this report we describe four steps in the purification of the kinase needed for release of membranes from chromatin. We also show that the release kinase and the mitotic initiation kinase, cdc2, are distinct and are separated from each other during the second purification step. Reconstitution experiments using these two kinases demonstrate that the release kinase and cdc2 kinase work in concert to cause membrane release from chromatin. In phosphorylation experiments, protein targets that are substrates for the regulatory release kinase are identified on the membranes. These phosphorylated proteins are candidates for regulated proteins mediating membrane-chromatin interaction. Finally, we find that membrane release activity can also be extracted from membranes by high salt treatment, indicating a possible dual localization of this activity.
Recent progress in understanding the dynamics of the cell nucleus has come predominantly from a biochemical approach employing in vitro systems that faithfully reproduce nuclear assembly and disassembly. The most widely used among these in vitro systems employs extracts prepared from mature Xenopuslaevis eggs(1, 2, 3, 4) . These cytoplasmic extracts can be prepared in either a mitotic or an interphase state. Mitotic extracts promote nuclear disassembly(5, 6, 7) , and interphase extracts support nuclear assembly around a DNA substrate(1, 3, 8) . Concerning assembly of the nuclear envelope membrane, a binding and a fusion step of precursor membrane vesicles to the nuclear envelope membrane has been resolved. It has been shown that binding of membrane vesicles to chromatin does not require cytosol, provided the chromatin DNA is present in a partially decondensed form(9, 10, 11) .
Using this in vitro binding assay, we have established that membrane binding to chromatin is regulated by the activities of a kinase and phosphatase system(9) . The kinase appears to phosphorylate a membrane bound component, which then inhibits association of vesicles with the chromatin. By contrast, the phosphatase appears to dephosphorylate the membrane component and promote binding of vesicles to chromatin. By modulating the relative activities of these two components in an assembly extract one can promote either vesicle-chromatin association or vesicle-chromatin dissociation.
At mitosis, chromatin detaches from the nuclear envelope in all eukaryotic cells. This dissociation occurs whether the cell vesicularizes its nuclear envelope (open mitosis) or the envelope remains intact during this period of the cell cycle (closed mitosis). This suggests that the relative activities of the kinase and phosphatase regulating membrane-chromatin association change at mitosis in favor of phosphorylation. In support of this we have shown that, although the major mitotic kinase, cdc2, is unable to cause membrane-chromatin dissociation directly, it can do so when acting in concert with the regulatory kinase contained in a crude cytosolic protein fraction(9) .
In this paper, we describe the partial purification of the membrane release activity. Starting from mitotic Xenopus cytosol, four steps of purification are described. The first column of the purification protocol, DEAE sepharose, led to a separation of all three regulatory activities, with cdc2 kinase eluting first, followed by the regulatory phosphatase and the regulatory kinase (measured as membrane release activity). The purest protein fraction containing membrane release activity also contained kinase activity phosphorylating a distinct set of membrane-associated proteins. Concerning the regulatory phosphatase, an assay was developed for purification of this activity. We also found that membrane release activity could be extracted from membranes by high salt treatment, indicating a possible dual localization of this regulatory enzyme, consistent with its proposed function. This is also supported by the observation that the kinase isolated from cytosol and the membrane-associated kinase phosphorylated a similar set of membrane proteins.
Mitotic cytosol-membrane fraction from
several preparations was thawed and pooled to give a total volume of 25
ml. 25 ml of EB buffer (80 mM -glycerophosphate, pH 7.3,
20 mM EGTA, 15 mM MgCl
, 1 mM dithiothreitol), 50 ml of 0.5 mM microcystin LR
(Calbiochem; 0.5 mM final concentration), and 250 ml of 50
mM ATP
S (to give a total final concentration of 0.5
mM) were added, and the membrane fraction was separated from
the cytosol fraction by centrifugation for 1 h at 100,000
g in a Beckman SW 41 rotor. The cytosol fraction was used for
ammonium sulfate precipitation while the membrane pellet was
resuspended in egg lysis buffer (250 mM sucrose, 50 mM KCl, 2.5 mM MgCl
, 10 mM Hepes/NaOH,
pH 7.4), frozen in liquid nitrogen, and stored at -70 °C.
The ammonium sulfate precipitate was dissolved in 10 ml of EB
buffer, 0.5 mM ATPS, and 0.5 mM microcystin LR.
30 ml of 10 mM Tris/HCl, pH 7.5, was added, and nondissolved
material was removed by centrifugation (clinical centrifuge). The
supernatant was applied, at a flow rate of 1 ml/min, to a
DEAE-Sepharose column (Pharmacia, 0.8
16 cm), equilibrated in
buffer A (20 mM
-glycerophosphate, 2.5 mM MgCl
, 5 mM EGTA, 1 mM
dithiothreitol, 20 mM Tris/HCl, pH 7.5). The column was
washed, at a flow rate of 2 ml/min, with buffer A, and protein was
eluted, at the same flow rate, with a 300-ml linear gradient
(0-0.5 M NaCl) in buffer A. Fractions of 6-ml volume
were collected, frozen, and kept at -70 °C. Membrane release
activity eluted between 300-400 mM NaCl.
In the very early stages of purification we realized that binding of membrane vesicles to chromatin was strongly ionic strength-dependent, and salt concentrations above 120 mM NaCl reduced dissociation of membranes from chromatin. To avoid ambiguous results due to differences in NaCl concentrations, all protein fractions assayed for membrane release were dialyzed against 100 volumes of EB buffer for 3 h prior to use.
We have previously characterized an experimental system to study the binding of membrane vesicles to demembranated Xenopus sperm chromatin in the absence of cytosolic components(9) . In this system, washed membrane vesicles, isolated from X. laevis eggs, are incubated with demembranated frog sperm chromatin in the presence of poly-L-glutamic acid. Under these conditions, the initially highly condensed sperm DNA becomes decondensed and then readily binds the purified membrane vesicles. The binding of membrane vesicles to chromatin can be easily visualized by fluorescence microscopy using dyes specific for either DNA or membranes.
Using this membrane-chromatin complex as a substrate, we have developed an assay for measuring the activity of the regulatory kinase contained in Xenopus egg cytosol. When interphase cytosol is added to this substrate, release of chromatin-bound membrane vesicles is observed provided that ATP and okadaic acid (an inhibitor of type 1 and 2A phosphatases) are present. Membrane release resulted in loss of fluorescent membrane stain on chromatin as assessed visually by fluorescence microscopy ( (9) and Fig.1). We have used this assay for purification of membrane release activity from Xenopus egg cytosol.
Figure 1: Membrane release activity in the heparin-Sepharose fraction. Membrane release activity was purified to the heparin-Sepharose fraction as described under ``Materials and Methods.'' 40-ml aliquots were employed in membrane release assays in the absence (Heparin Seph.Fraction) and presence (Heparin Seph. Fraction + DMAP) of the kinase inhibitor DMAP. Treatment of the samples with DMAP was as follows. In parallel, the substrate of membrane vesicles bound to chromatin and one aliquot of active fraction were incubated with 3 mM DMAP (added from a 0.3 M stock in dimethyl sulfoxide) for 30 min at room temperature. Control samples were treated identically in the absence of DMAP. Then, corresponding membrane-chromatin substrates and heparin-Sepharose fractions were combined, and membrane release activity was measured in the presence of an ATP-regenerating system as described under ``Materials and Methods.'' Heparin Seph., no kinase inhibitor added. Heparin Seph. + DMAP, kinase inhibitor added. DNA (leftpanels) and membrane stain (rightpanels) after 2.5 h of incubation are shown.
Mitotic egg cytosol was used as starting material for the purification because it was apparent from our previous investigations that membrane release activity is markedly enhanced in mitotic extracts relative to interphase extracts(9, 10) . In mitosis, the membrane-chromatin binding equilibrium is reversibly altered in favor of membrane release possibly due to mitotic stimulation of the regulatory kinase by cdc2 kinase. So far, we have established four purification steps for the release kinase including ammonium sulfate precipitation, chromatography on DEAE-Sepharose, Mono Q, and heparin-Sepharose (see Table1).
Fractionation of mitotic Xenopus cytosol
with 1.2 M (w/v) ammonium sulfate has previously been
described as yielding a crude preparation of maturation-promoting
factor/cdc2 kinase(12, 14, 18) .
Recently we found that this 1.2 M ammonium sulfate fraction
also contained high membrane release activity ( (9) and Table1). When cdc2 kinase was depleted from this crude
maturation-promoting factor preparation, membrane release activity was
still present. However, in contrast to the undepleted ammonium sulfate
fraction, it could now only be detected in the presence of okadaic
acid. ()Thus, it appears that in addition to cdc2 kinase both the regulatory kinase and the phosphatase are also
contained in this 1.2 M ammonium sulfate fraction.
Chromatography of the 1.2 M ammonium sulfate fraction on DEAE-Sepharose provided a means for separation of all three regulatory activities, with cdc2 kinase eluting at lowest ionic strength followed by the phosphatase (see below) and then the membrane release activity. At this point of purification the membrane release activity is partially contaminated with regulatory phosphatase since the phosphatase inhibitor okadaic acid (or microcystin LR, a phosphatase inhibitor with similar specificity that was used in many experiments) was required to detect membrane release activity. Phosphatase activity antagonizing membrane release was no longer observed in the active fractions of the following purification step (Mono Q column), which also provided concentration of the activity. Chromatography beyond the Mono Q fraction proved to be difficult because release activity became increasingly unstable. Fig.1shows release activity of the peak fraction of the last purification step established to date, chromatography on heparin-Sepharose CL-2B. Membrane release using this fraction was ATP-dependent and inhibited by 6,6`-dimethyl aminopurine (DMAP), a kinase inhibitor with broad specificity.
Following chromatography on a Mono Q column the protein content of fractions containing release activity was approximately 0.05% of the protein present in the starting cleared mitotic cytosol (Table1). However, the apparent purification at this stage seems rather low (40-100-fold). This low purification is largely due to the separation of cdc2 kinase, an activator of the release kinase activity, from the release kinase during DEAE chromatography. Following the separation of these two kinases the release kinase activity appears to attenuate. This attenuation in release kinase activity can be reversed by the addition of purified active cdc2 kinase (see below). Depending on the preparations used we found that the addition of purified cdc2 kinase to either the Mono Q or the heparin-Sepharose release kinase fractions increased the release activity present in these fractions 4-10-fold. If this normalization is incorporated into calculations for purification, then the purification achieved following Mono Q chromotography ranges between 160- and 400-fold.
Figure 2: Separation of histone H1 kinase and membrane release activity by chromatography on DEAE-Sepharose. Mitotic Xenopus egg cytosol was fractionated by ammonium sulfate precipitation as described under ``Materials and Methods.'' The resulting 1.2 M ammonium sulfate fraction was then applied to a DEAE column, and proteins were eluted with a 0-0.5 M NaCl gradient. The conditions for the DEAE chromatography were the same as described under ``Materials and Methods'' except that the volumes of mitotic cytosol, DEAE column, and NaCl gradient were only one-fourth of the respective volumes described there. Dashedline, NaCl gradient. Filledcircles, histone H1 kinase activity. Fractions from the DEAE column were employed at a 10-fold dilution in histone H1 kinase assays(9, 14) . As indicated by the bar, membrane release activity eluted in fractions 14-16.
Figure 3: Membrane release activity is stimulated by cdc2 kinase. Membrane release activity at the Mono Q stage was employed in membrane release assays in a 20-fold dilution, in the absence (Mono Q frc., 1:20 dilution) or presence of cdc2 kinase (Mono Q frc., 1:20 dilution, + cdc2 kinase). cdc2 kinase was affinity-purified by p13-Sepharose chromatography, as outlined under ``Materials and Methods,'' and 10 ml of cdc2 kinase eluted with p13 protein was used. In a control, the effect of cdc2 kinase on membrane binding in the absence of the Mono Q fraction was examined (No Mono Q frc., + cdc2 kinase). DNA stain (leftpanels) and membrane stain (rightpanels) are shown after 75 min of incubation.
Figure 4: Regulatory phosphatase restores membrane binding. Assays for the regulatory phosphatase were carried out as described under ``Materials and Methods.'' DEAE fractions able to restore binding eluted at about 0.25 M NaCl salt concentration, corresponding to fractions 12 and 13 shown in Fig.2. 20 ml of active DEAE fraction, in the absence (DEAE fraction) or presence of the phosphatase inhibitor microcystin LR (DEAE fraction + Microcystin LR) were used in the assay. DNA stain (leftpanels) and membrane stain (rightpanels) after 2 h of incubation are shown.
Figure 5:
Phosphorylation of membrane-associated
proteins by membrane release kinase. A, membranes purified
from X.laevis extracts were used in kinase assays,
in the absence (lanes1-5) or presence (lanes6-10) of Mono Q fraction of membrane
release activity (10-fold diluted), as described under ``Materials
and Methods.'' Assays were carried out with varying amounts of
membranes, as indicated in the figure. After incubation for 15
min at room temperature, membranes were sedimented to terminate
phosphorylation. An autoradiograph of P-labeled proteins,
recovered in the membrane pellets and resolved by SDS-gel
electrophoresis, is shown. B, Mono Q fraction of membrane
release kinase (3 ml per sample) was assayed for kinase activity, in
the absence (lane1) or presence (lane2) of 0.5 mg of membranes, as described under
``Materials and Methods.'' In this experiment, however, the
total sample was analyzed for phosphorylated proteins without
separating membranes from soluble proteins. C, kinase assays
were as described in B using 3 ml of Mono Q fraction of
membrane release kinase/sample. Membranes were present in the amounts
indicated in the figure. After incubation for 15 min at room
temperature, samples were mixed with 40 ml of 2.4 M sucrose.
0.3 ml of 1.2 M sucrose in EB buffer and 50 ml of EB were
layered on top to create a sucrose step gradient. The samples were
centrifuged for 1 h at 45,000 rpm in a Beckman TLS-55 rotor. 0.2 ml of
the top portion of the gradient, containing flotated membranes, were
removed and diluted with 1 ml of EB buffer, and membranes were
sedimented by centrifugation for 1 h at 45,000 rpm in a Beckman
TLA-100.3 rotor. Phosphorylated proteins in the pellet were analyzed by
SDS-gel electrophoresis and
autoradiography.
In a
second experiment, the kinase activity in the active Mono Q fraction
was assessed. The Mono Q fraction was incubated with
[-
P]ATP in the absence or presence of
limiting amounts of membranes that by themselves would cause only
background phosphorylation (see Fig.5A, lane1). In contrast to the experiment described above, the
total samples were analyzed for phosphorylated proteins. Kinase
activity in the Mono Q fraction led to phosphorylation of only a
limited number of proteins (Fig.5B, lane1). However, when membranes were present, overall
phosphorylation was considerably increased (Fig.5B, lane2). It was striking that some of the
phosphorylated protein species appeared to be identical, regardless of
whether membranes were present or not. Together with the result from Fig.5A, this indicates that some of the target
proteins for membrane release kinase display dual localization, either
soluble or membrane-bound.
The observation of a dual localization of some phosphorylated proteins raised the question of which of the target proteins were actually membrane-associated. To determine which of the phosphorylated proteins were truly membrane associated we performed an experiment in which membranes were first phosphorylated with membrane release kinase and then the membranes were recovered by flotation (Fig.5C). Under these conditions, we observed that the majority of the target proteins, including those contained initially in the Mono Q fraction, now cofractionated with the membranes, indicating they were truly membrane-associated proteins. Taken together we conclude from these data, that membrane release kinase as well as its target proteins can be located in the cytosol as well as on the membrane vesicles.
Figure 6: Membrane release activity can be extracted from membranes. Membranes isolated from Xenopus egg extracts were extracted with 1 M KCl as described under ``Materials and Methods.'' 25-ml aliquots of the salt extract, dialyzed into EB buffer, were employed in membrane release assays as described under ``Materials and Methods.'' The salt extract was assayed for membrane release without (KCl extract) or with kinase inhibitor (KCl extract + DMAP). Treatment with DMAP (3 mM final concentration) was as described in Fig.1. DNA (leftpanels) and membrane stain (rightpanels) after 1 h of incubation are shown.
In higher eukaryotes, a very early step in the nuclear
assembly pathway at the end of mitosis is the binding of membrane
vesicles on the chromatin surface. We previously described an in
vitro binding assay to study this particular step in nuclear
reconstitution in the absence of nuclear pore complex formation and
lamin assembly (9) . We found this binding assay applicable
with chromatin from different sources(10, 11) ,
indicating a generally conserved mechanism for the interaction of
chromatin with membranes. The binding observed in this assay defines an
intermediate stage of nuclear assembly that can be completed by adding
back the cytosolic fraction from Xenopus extracts(10, 11) . Furthermore, the
membrane-chromatin interaction is protein-mediated, and mild treatment
of the membranes with protease abolishes binding ((10) ), and, consequently, nuclear assembly(8) . In this study we
utilized this assay to develop protocols with the aim to identify the
enzymes that regulate the membrane-chromatin interaction as well as to
identify the protein components that mediate it. We have established
assays for the purification of a regulatory protein kinase and its
counterpart, a protein phosphatase. While the kinase promotes release
of chromatin-bound membrane vesicles and, therefore, appears to be a
key enzyme in the disassembly of the nuclear envelope membrane,
activity of the phosphatase restores binding and, consequently, is
required for assembly of the nuclear envelope membrane. We also used an
established procedure to purify cdc2 kinase, an activator of
membrane release in mitosis (9) and found that one pathway of
membrane release activation involves the combined action of cdc2 kinase and membrane release kinase.
On the basis of active fractions being able to cause membrane release, four steps of purification for the regulatory kinase have been established starting from mitotic cytosol. Separation of the kinase from the regulatory phosphatase, as well as from cdc2 kinase, has been achieved. An assay and a crude form of purification for the regulatory phosphatase have been established, which will be the basis for a further purification of this activity. Using a kinase assay to identify possible targets for the regulatory kinase we found that a distinct set of membrane-associated proteins is phosphorylated by membrane release kinase. Kinase activity with similar substrate specificity was also found associated with the membrane vesicles. Accordingly, membrane release activity could be extracted from membranes by high salt treatment, making a dual localization of membrane release kinase possible. Surprisingly, some of the membrane-associated target proteins were present as soluble species in partially purified fractions of the kinase and were phosphorylated in the absence of membranes. It therefore appears that the regulatory kinase as well as substrates phosphorylated by this kinase can be localized on membranes as well as in the cytosol.
It has been previously observed that affinity-purified cdc2 kinase stimulates membrane release activity(9) . In this report we have shown that in the absence of the regulatory phosphatase addition of cdc2 kinase increases both membrane release activity and the kinase activity present in this fraction. At this stage, without knowing the relevant substrates, we cannot unequivocally determine if the stimulatory effect results from activation of the regulatory kinase by cdc2 kinase or by both kinases phosphorylating a common substrate. In kinase assays, using a combination of cdc2 kinase and membrane release kinase, we found stimulation of phosphorylation of several membrane-associated target proteins, which will be subject to further analysis. We do not exclude the possibility that cdc2 kinase has an inhibitory effect on the regulatory phosphatase.
Membrane-associated proteins that are phosphorylated by membrane release kinase are our major candidates for the protein(s) mediating membrane binding to chromatin. The finding of a dual localization of target proteins for membrane release kinase raises intriguing questions about the mechanism of membrane release. Our previous hypothesis favored a model whereby binding is mediated by an integral membrane protein whose interaction with chromatin was dependent on its state of phosphorylation(9) . Currently available data are still compatible with the view that direct contact to chromatin is made by an integral membrane protein since high salt treatment of membranes does not abolish membrane binding. However, considering the results discussed above, we can not exclude the possibility of a more complicated regulatory mechanism whereby proteins required for membrane release can shuttle between cytosol and membranes and, depending on their state of phosphorylation, mediate dissociation of membranes from chromatin.