©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Assembly/Disassembly of the Nuclear Envelope Membrane
CHARACTERIZATION OF THE MEMBRANE-CHROMATIN INTERACTION USING PARTIALLY PURIFIED REGULATORY ENZYMES (*)

(Received for publication, May 5, 1995; and in revised form, June 6, 1995)

Rupert Pfaller John W. Newport (§)

From theDepartment of Biology, University of California, San Diego, La Jolla, California 92093-0347

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


MATERIALS AND METHODS

Fractionation of X. laevis Eggs

Published procedures were used to obtain interphase extracts (3, 8, 9) and mitotic extracts (7, 9, 12) from mature, unfertilized Xenopus eggs. Cytosol and membrane fractions were obtained from crude egg extracts by ultracentrifugation (1 h at 55,000 rpm, Beckman TLS-55 rotor) as described previously(9) .

Purification of Membrane Release Activity

Preparation of Mitotic Cytosol for Protein Purification

Mitotic cytosol for purification of membrane release activity was prepared the following way. After ultracentrifugation of crude mitotic egg extract, the resulting cytosol layer together with the membrane layer were separated from a bottom pellet consisting predominantly of glycogen and residual cellular debris. ATPS (^1)(Boehringer Mannheim) was added (0.5 mM final concentration), and aliquots of the membrane-cytosol mixture (1-ml volume) were frozen in liquid nitrogen and stored at -80 °C until use.

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 beta-glycerophosphate, pH 7.3, 20 mM EGTA, 15 mM MgCl(2), 1 mM dithiothreitol), 50 ml of 0.5 mM microcystin LR (Calbiochem; 0.5 mM final concentration), and 250 ml of 50 mM ATPS (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(2), 10 mM Hepes/NaOH, pH 7.4), frozen in liquid nitrogen, and stored at -70 °C.

Ammonium Sulfate Precipitation

Ammonium sulfate precipitation of mitotic cytosol was essentially carried out as described for the partial purification of maturation-promoting factor(12, 18) . The volume of mitotic cytosol was adjusted to 50 ml with EB buffer, and 25 ml of 3.6 M ammonium sulfate, dissolved in EB, was added dropwise with frequent mixing (1.2 M final concentration). After incubation for 30 min on ice, precipitated protein was recovered by centrifugation (Sorvall HB-4 rotor, 10 min at 10,000 rpm).

DEAE-Sepharose Chromatography

All chromatographic procedures described in the following sections were carried out using fast protein liquid chromatography (Pharmacia Biotech Inc.) in the cold room.

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 beta-glycerophosphate, 2.5 mM MgCl(2), 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.

Mono Q Chromatography

Active fractions from the DEAE column were pooled (total volume, 48 ml), microcystin LR was added (0.5 mM final concentration), and the pool was divided in half, both of which were run over a Mono Q column (Pharmacia) under identical conditions. 24 ml of DEAE pool was diluted with 26 ml of buffer A and, using a 50-ml super loop (Pharmacia), loaded onto a Mono Q column (Pharmacia; flow rate 1 ml/min, equilibrated in buffer A, 0.1 M NaCl). The column was washed with 10 ml of equilibration buffer, and protein was eluted, at a flow rate of 0.5 ml/min, with a 25-ml linear gradient (0.2-0.5 M NaCl) in buffer A. Fractions of 1-ml volume were collected, frozen in liquid nitrogen, and stored at -70 °C. Membrane release activity eluted as a sharp peak at about 380 mM NaCl. Both Mono Q runs were combined, and 4 ml of active Mono Q fraction were recovered.

Heparin-Sepharose CL-2B Chromatography

1 ml of active Mono Q fraction was diluted with 3 ml of buffer A and, using a 10-ml super loop (Pharmacia), was loaded at a flow rate of 0.4 ml/min onto a heparin-Sepharose CL-2B column (Pharmacia HR 5/20, 5 200 mm), equilibrated in buffer A (heparin-Sepharose CL-2B was a generous gift from Jim Kadonaga, UCSD). The column was washed with 6 ml of buffer A, and protein was eluted by a two-step salt gradient, 4 ml of a gradient from 0-180 mM NaCl followed by 30 ml of a gradient from 180-600 mM NaCl, both in buffer A. 1-ml fractions were collected, frozen in liquid nitrogen, and stored at -70 °C. Membrane release activity eluted at about 480 mM NaCl. At this stage, the protein concentration of the active fraction was very low, and membrane release activity became sensitive to freezing and thawing.

Assay for Membrane Release Activity

An assay, modified from our previously described protocol(9) , was used to measure membrane release activity.

Membrane Binding to Demembranated Frog Sperm Chromatin

In a first step, the substrate for membrane release assays was prepared in a mix, here described for 10 assays. To 0.1 ml of egg lysis buffer, containing 2 mg of poly-L-glutamic acid/ml (Sigma), membrane vesicles (30 mg, usually from a 5 mg/ml stock), demembranated frog sperm chromatin (7,500/ml, isolated from the testes of male X. laevis frogs as described in Refs. 1 and 8), ATP (2 mM), GTPS (0.2 mM), and aprotinin and leupeptin (100 mg/ml each) were added and incubated for 30 min at room temperature. Although not required, for most of the membrane release assays and all of the kinase assays (see below) membranes isolated from Xenopus egg extracts were further purified by flotation(8) . Protein concentrations of membrane suspensions (and also of fractions from the protein purification) were determined using the Bradford assay (Bio-Rad).

Membrane Release

10-ml aliquots of the binding mix were added to 40 ml of protein fraction in EB buffer and 5 ml of a 10-fold concentrated ATP-regenerating mix (consisting of 0.5 mg of creatine kinase/ml, 10 mM ATP, 50 mM creatine phosphate), and, if required, okadaic acid (Moana Bioproducts) or microcystin LR (1 mM final concentration of either phosphatase inhibitor) was added. Binding of membrane vesicles to chromatin was assessed by fluorescence microscopy as described previously (9) using a combination of the fluorescent dyes Hoechst 33258 (DNA) and 3,3`-dihexyloxacarbocyanine (Kodak) (membranes).

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.

Assay for the Regulatory Phosphatase

Based on our previous observation(9) , in a first step, membranes isolated from Xenopus interphase extracts were inactivated for chromatin binding by incubating 3 ml of membrane stock for 30 min at room temperature in 30 ml of interphase cytosol, containing an ATP-regenerating system, 2 mM GTPS, and 2.5 mM microcystin LR. The incubation mixture was then layered on top of 50 ml of egg lysis buffer containing 0.5 M sucrose, the membranes were reisolated by centrifugation (Eppendorf centrifuge, 15 min at 4 °C), and the membrane pellet was resuspended in 30 ml of lysis buffer. In parallel, a mix was prepared consisting of 0.1 ml of EB buffer containing 2 mg of poly L-glutamic acid/ml, 1 mM ATP, and demembranated frog sperm chromatin (7,500 sperm/ml). After incubation for 20 min at room temperature, 15 ml of pretreated membranes were added, and 20-ml aliquots of the resulting mix combined with 20 ml of phosphatase-containing protein fraction (dialyzed into EB buffer as described above). Aliquots of the samples were assessed for restoration of membrane binding to chromatin by fluorescence microscopy as described above. In control samples, microcystin LR was added (2 mM final concentration) to inhibit phosphatase activity.

Phosphorylation of Membrane-associated Proteins

Protein fractions to be assayed for kinase activity were adjusted to a final volume of 10 ml using EB and 10 ml of a mix was added containing 0.4 mM ATP, 2 mM microcystin LR, 5-10 mCi of [-P]ATP (ICN), and, if not otherwise indicated, 0.5 mg of membranes isolated from Xenopus egg extracts (further purified by flotation; see membrane release assay). Samples were incubated for 15 min at room temperature and diluted with 200 ml of EB buffer, and membranes were reisolated by centrifugation (Eppendorf centrifuge; 12 min at 4 °C). The membrane pellets were dissolved in SDS-containing sample buffer and analyzed by SDS-polyacrylamide gel electrophoresis (13) and autoradiography of the dried gels. If phosphorylation of the total sample was analyzed, 7 ml of 4-fold concentrated SDS-containing sample buffer was added to stop the kinase reaction.

Affinity Chromatography of cdc2 Kinase on p13-Sepharose

Fractions from the DEAE column were assayed for histone H1 kinase activity as described previously(9, 14) . Active fractions were pooled, and one-half of the pool (25-ml volume) was applied to a 1.5-ml column of p13-Sepharose. p13-Sepharose was prepared by coupling purified, recombinant p13 protein (15) to CNBr-activated Sepharose (Pharmacia) at a concentration of 10 mg of p13/ml of swollen gel, following the manufacturer's instructions. After loading, the column was washed with 20 ml of buffer A, 0.2 M NaCl followed by 15 ml of buffer B (1 mM EGTA, 1 mM dithiothreitol, 50 mM Tris/HCl, pH 6.8). cdc2 kinase was then eluted with 2 ml of 30 mg/ml p13 in buffer B(17) . 1-ml fractions were collected, and aliquots were assayed for histone H1 kinase activity as described(9, 14) . Active fractions were frozen in liquid nitrogen and stored at -70 °C.

1 M KCl Extraction of Xenopus Membranes

A pool of 1.8 ml of membrane suspension, derived from several preparations of interphase extract by ultracentrifugation of the crude extract (approximately 18-ml volume), were diluted with EB buffer to a final volume of 10 ml. Aprotinin and leupeptin were added (10 mg/ml final concentration), and the membranes were reisolated by centrifugation in a Beckman TLA-100.3 rotor (1 h, 50,000 rpm). The volume of the firm membrane pellet was determined to be 0.75 ml. It was suspended in a total volume of 1 ml (by adding EB), and then 1 ml of 3-fold concentrated EB, 1 ml of 3 M KCl (1 M final concentration), 1 mM ATP, aprotinin, and leupeptin (20 mg/ml final concentration each) were added. The membranes were thoroughly suspended and kept at room temperature for 15 min followed by centrifugation in a TLA-100.3 rotor (1 h at 60,000 rpm). The supernatant (2.2-ml volume) was dialyzed against 250 ml of EB for 3 h with one change of the dialysis buffer after 1 h. Aliquots of the salt extract were frozen in liquid nitrogen and kept at -70 °C.


RESULTS

Purification of Membrane Release Activity

Mature X. laevis eggs can be fractionated by centrifugation. Independent membrane and cytosol fractions can be obtained from such a fractionation, which together will support assembly of a functionally and morphologically intact nuclear envelope around a chromatin substrate in vitro(3, 8) . Modification of this system has opened the way to study the specific binding of membrane vesicles on the chromatin surface (9, 10, 16) and their subsequent fusion to form the double membrane of the nuclear envelope (10, 11) .

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. (^2)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.

Stimulation of Membrane Release Activity with cdc2 Kinase

As previously demonstrated, cdc2 kinase is able to stimulate membrane release(9) . Since separation of the regulatory enzymes was not available at that time, it was not clear if stimulation of membrane release was caused by stimulation of the kinase or attenuation of the phosphatase. As shown in Fig.2, chromatography of the 1.2 M ammonium sulfate cut on DEAE-Sepharose separated cdc2 kinase activity (assayed by phosphorylation of histone H1, one of its substrates) and membrane release activity. Accordingly, we observed a sharp drop in membrane release activity (Table1) and a concomitant dependence of membrane release on okadaic acid. To answer the question of whether cdc2 kinase stimulates membrane release activity in concert with the regulatory kinase, cdc2 kinase was further purified by affinity chromatography on p13 Sepharose (17) and assayed in combination with membrane release activity at the Mono Q stage. As already mentioned, at this stage of purification, regulatory kinase and phosphatase appeared to have been separated completely. The stimulatory effect of cdc2 kinase on membrane release was assayed the following way. Active Mono Q fraction was employed in the membrane release assay at a 20-fold dilution, in the absence or presence of cdc2 kinase. At this dilution, the Mono Q fraction alone failed to cause membrane release (Fig.3, Mono Q frc., 1:20 dilution). Membrane release was restored, however, when cdc2 kinase was added (Fig.3, Mono Q frc., 1:20 dilution, + cdc2 kinase). cdc2 kinase alone did not lead to membrane release even when present at a 2-fold higher concentration. Thus, in this particular case, membrane release activity was stimulated about 4-fold in the presence of cdc2 kinase. It should be noted that stimulation of membrane release varied considerably depending on the preparation of membrane release kinase, and stimulation up to 10-fold has been observed. The cdc2 kinase-mediated activation of membrane release in the absence of the regulatory phosphatase indicates that one pathway of stimulating membrane release in mitotic extracts is mediated by the combined effect of a regulatory kinase and cdc2 kinase. To date, neither the regulatory kinase nor its substrate(s) have been identified and, although we favor a mechanism by which stimulation of membrane release occurs in a linear fashion where cdc2 kinase stimulates the activity of the regulatory kinase, we cannot exclude the possibility that both kinases act in parallel (see ``Discussion'').


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.



The Regulatory Phosphatase

When isolated membrane vesicles are incubated in interphase cytosol in the presence of phosphatase inhibitors (either okadaic acid or microcystin LR) and then isolated by centrifugation they will no longer bind to sperm chromatin(9) . This inactivation of binding is dependent on release kinase and likely involves phosphorylation of membrane proteins (see below). We have used membrane vesicles, pretreated with interphase cytosol and phosphatase inhibitors, as a substrate to purify the regulatory phosphatase that would restore binding to inactive vesicles. Assaying fractions from the DEAE-Sepharose column for fractions that would restore binding to inactivated vesicles, we found that phosphatase activity eluted between the peaks of cdc2 kinase and membrane release activity. As shown in Fig.4, restoration of membrane binding was sensitive to microcystin LR, indicating that the activity of a phosphatase 1 or 2A in the DEAE fraction was required to restore binding. In summary, this approach constitutes a simple purification assay for the regulatory phosphatase.


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.



Kinase Activity Associated with Membrane Release Activity

What are the target proteins of the regulatory kinase? Our work has indicated that substrate proteins for the regulatory kinase are localized on the membrane vesicles. Among these target proteins we expect to find the regulated protein(s) mediating membrane-chromatin interaction. To investigate this further we developed a kinase assay using membrane vesicles, purified by flotation (8) , as substrate for phosphorylation. These membranes were incubated with release kinase fractions in the presence of [-P]ATP. Following this labeling, phosphorylated proteins associated with membranes were separated from soluble proteins by centrifugation and analyzed by SDS-gel electrophoresis and autoradiography. Because the membranes themselves contain endogenous kinase we first compared the phosphorylation patterns of different amounts of membranes incubated in the presence and absence of the release kinase purified through the Mono Q step (Fig.5). Results from these experiments showed that a distinct set of membrane-associated proteins are phosphorylated by the release kinase. At a fixed kinase concentration the amount of each of these protein phosphorylations increased linearly with increased membrane concentration. Interestingly, at high membrane concentrations in the absence of added release kinase, proteins phosphorylated by endogenous membrane kinase were very similar to those observed in the presence of release kinase (Fig.5A, compare lanes5 and 10). This indicates that a small amount of release kinase may be bound to the membranes (see below). However, despite this similarity it is clear that addition of release kinase purified through the Mono Q step stimulated phosphorylation of these proteins approximately 5-fold over endogenous membrane kinase levels.


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.

Release Activity Can Be Extracted from Membranes by High Salt Treatment

In the previous section we presented data that pointed to a dual localization of membrane release activity in the cytosol as well as on membranes. To directly address this question, we prepared extracts from membranes by high salt treatment. After dialysis into assay buffer, the salt extract was employed instead of cytosolic fractions in the membrane release assay. Membrane release activity in the salt extract was indeed observed when ATP and okadaic acid were present (Fig.6). Moreover, membrane release was inhibited by the kinase inhibitor DMAP. Simple dissociation of membranes from chromatin due to insufficient dialysis can be excluded because the observed activity in the salt extract was heat-labile (not shown). Thus, membrane release activity derived from cytosol and membrane release activity derived from membranes display the same characteristics. Further purification will be required to answer the question of whether both kinases are actually identical. It should be noted that dependence of membrane release on okadaic acid in salt extracts was batch-dependent, and some extracts were obtained where no phosphatase inhibitor was required in membrane release experiments. The requirement for phosphatase inhibitors indicates that not only the regulatory kinase but also the regulatory phosphatase can be membrane-associated.


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.




DISCUSSION

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) ),^2 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.

Separation of the Regulatory Enzymes That Determine the Membrane-chromatin Binding Equilibrium

After heparin-Sepharose chromatography, the last step of purification of the membrane release kinase, only a few proteins were present in the active fraction (based on silver staining of gels). The protein concentration dropped considerably following this step, and the activity became unstable, which made a further purification difficult. Among the remaining protein species there was no obvious candidate for the regulatory kinase based on molecular weight. Moreover, specific inhibitors or activators of known kinases (for example protein kinases A and C or calmodulin-dependent protein kinase) had no effect when they were employed in membrane release or kinase assays using this purest kinase fraction.^2 However, membrane release activity, at any stage of purification, was ATP-dependent and sensitive to DMAP, a kinase inhibitor of broad specificity, emphasizing that a kinase is the enzymatic activity central to membrane release. This conclusion is supported by a comparison of membrane release activity and kinase activity at the different stages of purification using Xenopus membranes as substrate. After chromatography on DEAE-Sepharose two peaks of gross kinase activity were observed. The kinase peak eluting at higher salt concentration contained membrane release activity. During subsequent purification of this kinase activity via chromatography on Mono Q and heparin-Sepharose, this kinase peak continued to purify as though it were a single component, i.e. multiple kinase peaks were not observed. Importantly, in each case, membrane release activity co-fractionated with kinase activity. This strongly indicates that membrane release and kinase activity are part of the same component. Therefore, it appears that the more sensitive kinase assay can be used as a reliable diagnostic tool in the purification of membrane release activity. Upon gel filtration, membrane release activity and kinase activity eluted with an approximate molecular weight of 150-200 kDa, making it possible that the regulatory kinase is organized in a protein complex. (^3)

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.

Double Localization of Membrane Release Activity and Its Protein Targets

In phosphorylation experiments we found kinase activity associated with membrane vesicles. Surprisingly, the protein targets phosphorylated by the membrane-associated kinase and by membrane release kinase were almost identical. However, in the presence of membrane release kinase a marked stimulation of membrane associated phosphorylation was observed (Fig.5A). Moreover, it appears that some of the phosphorylated target proteins were present on the membranes and soluble in fractions of the membrane release kinase (Fig.5B). Whether the membrane-associated kinase and membrane release kinase are identical has yet to be established; however, considering the very similar substrate specificity, a dual localization of membrane release kinase appears to be likely. This becomes more likely in light of the observation that membrane release activity with characteristics of the one purified from cytosol can be extracted in an active and soluble form from membranes by high salt treatment.

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.


FOOTNOTES

*
This work was supported by a fellowship from the Deutsche Forschungsgemeinschaft (to R. P.) and by National Institutes of Health Grant GM 33523 (to J. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biology, 0347, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0347. Tel.: 619-534-3423; Fax: 619-534-0555.

^1
The abbreviations used are: ATPS, adenosine 5`-3-O-(thio)triphosphate; GTPS, guanosine 5`-3-O-(thio)triphosphate; DMAP, 6,6`-dimethyl aminopurine.

^2
R. Pfaller and J. Newport, unpublished observations.

^3
R. Pfaller and J. Newport, unpublished results.


ACKNOWLEDGEMENTS

We greatly appreciate the help from the Kadonaga lab and the Malhotra lab at UCSD who generously shared lab equipment during the course of this work.


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