Neurochem. Int. Vol. 21, No. 3, pp. 409--414,1992

0197-0186/92$5.00+ 0.00 Copyright © 1992PergamonPress Ltd

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GTP-BINDING PROTEINS IN BOVINE BRAIN N U C L E A R MEMBRANES* HENNING OTTO, KLAUS BUCHNER, ROLAND BECKMANN, RALF HILBERT a n d FERDINAND HUCHOt Arbeitsgruppe Neurochemie, Institut fiir Biochemie, Freie Universit~.t Berlin, Thielallee 63, 1000 Berlin 33, Germany (Received 21 November 1991 ; accepted 26 February 1992)

AbstractmNuclear membranes and other subcellular fractions derived from bovine brain cortex were investigated for the existence of GTP-binding proteins. By using photolytic labeling with [~-32P]GTPa 29 kDa GTP-binding protein was shown to be present in nuclear membranes which was not present in the plasma membranes nor in microsomal or cytosolic fractions. Two-dimensional gel electrophoresis revealed that this protein is rather acidic with a pl lower than 4.5. Members of the heterotrimeric Gi/ofamily are not present in the nuclear envelope: a 39 kDa protein, ADP ribosylated by pertussis toxin, was shown to originate from plasma membrane contamination.

For the regulation of many crucial cellular events as e.g. proliferation or differentiation it is necessary that signals are transmitted from the cell surface to the cell nucleus. The signalling pathways, which begin for example with the binding of growth factors to their receptors and end in altered gene expression, are poorly understood (for review see Nigg, 1990). As one step in the sequence of events a signal has to cross the nuclear envelope. In this context the transport of macromolecules through pore complexes and the pore complex itself has been the object of intensive investigations (Silver, 1991; Gerace and Burke, 1988). Recently, however, evidence was presented that shows that besides transport through pore complexes other processes take place at the nuclear envelope, which may play a role in signal transduction events. Examples are reports on inositol trisphosphate (IP3)sensitive Ca 2+ pools in nuclei (Nicotera et al., 1990) and on K+-channels (Mazzanti et al., 1990) and C I - channels (Tabares et al., 1991) in nuclear envelopes. One important class of proteins involved in signal transducing processes are GTP-binding proteins. One group of these proteins comprises the heterotrimeric G-proteins, which are essential for the signal trans-

duction through the plasma membrane by coupling transmembrane receptors to their intracellular effectors (for a recent review see e.g. Taylor, 1990). Another rapidly growing group are monomeric GTP-binding proteins of lower molecular weight, which appear to have important regulatory functions. The members of the ras superfamily are involved in proliferation control and are important components of the signaling pathways regulating intracellular vesicle transport (Balch, 1990; Hall, 1990). Concerning the nuclear envelope there were only some speculations on the existence of G-proteins in nuclear envelopes (Russell, 1989), but no thorough investigations on this possibility exist. On the other hand, GTP-binding proteins, as yet not fully characterized, were described to be present in liver nuclear envelopes (Takeda et aL, 1989; Rubins et al., 1990; Seydel and Gerace, 1991). The aim of this study was to search for GTP-binding proteins in nuclear envelopes of brain nuclei.

EXPERIMENTAL PROCEDURES

Materials

*This paper is dedicated to the memory of Professor D. Biesold who died on the 29 May 1991 at the age of 65. Dr Volker Bigl, long time colleague and friend of Dietmar Biesold acted as Executive Editor in the processing of this paper. TAuthor to whom all correspondence should be addressed. 409

[Adenylate 32P]NAD and [,,-32P]GTP were from NEN. DNase I was from Boehringer, Mannheim. Pertussis toxin, heparin and other chemicals were from Sigma. Subcellular fractionation

Nuclei, plasma membranes, microsomes and cytosoi were prepared from bovine brain cortex by modified procedures

410

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according to Thompson (t987), Jones e: oi, (1974) and Cotman et al. (1971). All steps were carried out at 4 C. After removing white matter, meninges and arteries, the cortex was cut into small pieces and homogenized in NP 0.32 (20 mM Hepes, pH 7.4, l mM MgCI:, 0.32 M sucrose, 1 mM PMSE, 10 lLg/ml leupeptin, I0 #g/ml aprotinin and 42.8 mM flmercaptoethanol) by 5 complete up and down strokes with a Teflon-glass homogenizer at 700 rpm. Following filtration through 110 mesh nylon gauze, the homogenate was made up to 15% (w/v) cortex. The homogenate was centrifuged at 170 g for 15 min. The resulting supernatant (S l) was further used for preparation of plasma membranes, microsomes and the cytosolic fraction. The pellet was resuspended in NP 0,32 and centrifuged at 370 g for 15 rain. Again, the pellet was resuspended in a small volume of NP 0.32 and adjusted to 2.0 M sucrose by addition of NP 2.4 (20 mM Hepes, pH 7.4, 2.4 M sucrose. 1 mM MgCI:). This suspension was centrifuged for 60 rain at 67,000g in a swinging bucket rotor (TST 28.38). After discarding the supernatant the pellet was carefully resuspended in a small volume of NP 0.32. An appropriate volume of NP 2.4 was added to obtain a concentration of 2.1 M sucrose. This suspension was transferred to a centrifugation tube and underlaid with NP 2.15 (20 mM Hepes, pH 7.4, 2.15 M sucrose, 1 mM MgClz). After centrifugation at 67,000 g for 60 rain the pellet was resuspended in a small volume o f N P 0.32. Integrity and purity of the nuclei were judged by phase contrast microscopy and by assaying 5'-nucleotidase (Kai et a/., 1966) as a plasma membrane marker. For preparation of plasma membranes, microsomes and cytosolic fraction the supernatant S1 was centrifuged at 8000 g for 15 min to obtain a crude membrane fraction (pellet P2) and the supernatant $2. The pellet P2 was resuspended in NP (20 mM Hepes, pH 8.1, I m M MgCI:, 1 rnM /~-mercaptoethanol, 10/~g/ml leupeptin, 10/~g/ml aprotinin, 1 mM PMSF) by 6 strokes in a glass-Teflon homogenizer (1000 rpm) and then incubated at 0 C for 60 rain. The lysate was made up to 1.05 M sucrose by addition of an appropriate volume of 20 mM Hepes, pH 8.1, 1.5 M sucrose, I mM MgCI_,, t mM /3-mercaptoethanol. The protein concentration was adjusted to 0.5 mg/ml. Of this suspension 15 ml was laid between 15 ml 20 m M Hepes, pH 7,4, 0.83 M sucrose, 1 mM MgCt> l mM ,6-mercaptoethanol and 4 ml NP 0.32. The density gradients were centrifuged at 105,000 g for 40 min in a swinging bucket rotor. The interface between 1.05 and 0,83 M sucrose was carefully removed, diluted with 2 vol o f N P and sedimented at 20,000g for 20 min. The pellet (highly purified plasma membranes) was resuspended in a small volume of NP 0.25. For the preparation of microsomes and the cytosolic fraction, the supernatant $2 was centrifuged at 48,700 g for 30 min. The resulting supernatant was diluted with N P to a concentration of 0.25 M sucrose and centrifuged at 105,000 g for 12 h. The microsomal pellet was resuspended in NP 0.25. The high speed supernatant was designated as cytosolic fraction. All fractions were stored at - 7 0 ° C

Preparation of nuclear era,elopes Nuclear membranes were prepared by a modification of the method o f Riedel and Fasold (1987). Nuclei (5 to 15 mg protein) were suspended in 200 ml TP buffer (10 mM TrisHCI pH 8.0, 10 mM Na,,HPO4). After adding 50 mg heparin and 2 mg DNase 1 (ca. 6000 U) the nuclei were incubated for 60 rain at 4°C and 15 min at room temperature with

gentle agitation. The suspension was then filtered through a nylon gauze (30 fun) and centrifuged at 9250 g t\~r t0 rain (4 C ) The pellet was washed once in NP 0 32 and le>us pended in a small volume of NP 0.32. Protein determination was pertormed according to Bradlord (1976) using bovine serum albumin (BSA/as standard.

A D P ribos.t'lalion with perlussis lo vin A D P ribosylation of G-proteins by pertussis toxin was performed according to Ribeiro-Neto et al. (1985). Briefly, the reaction was carried out in a total volume of 60 Id, containing protein of different sources at a concentration of 0.33 mg/ml (20 jug), 60 mM Na" Hepes, pH 7.4, 30 mM thymidine, 3 mM ATP. 0.3 mM GTP, 3 mM EDTA, 5 ill of activated toxin solution. [00 ~tM dithioerythritol (DTE) and 10 /~M [adenytate ~2P]NAD (2.3 /~Ci, 3.8 Ci/ lnmol). The reaction was started by the addition of [::P]NAD and DTE. After an incubation for 45 min at 32 C, the reaction was terminated by precipitation of the protein either by trichloroacetic acid (TCA) or methanol/chloroform The resulting pellets were prepared for S D S - P A G E

Photolytic labeling with [~-":P]GTP Photolytic labeling of GTP-binding proteins by [~32P]GTP was performed as described by Schleicher et at. (1986). A total volume o f 50 Id contained 0.4 mg/ml protein (20 pg), 40 mM Na" Hepes, pH 7.5, 0.4% Lubrol PX, 4 mM EDTA, 2 mM DTE and 10 nM [:~A:P]GTP (1,5/tCi, 3000 Ci/mmol). All components were mixed and preincubated for 30 min on ice. Then, the mixture was irradiated for 20 rain at room temperature by u.v.-light (0.35 W, 254 nm, distance : 15 cm). Following irradiation, the mixture was prepared for SDS-PAGE. In case of double labeling, the samples were first subjected to photolytic labeling by GTP. As a second step, A D P ribosytation was performed in a total volume o f t00 ld under the conditions described above.

Electrophoresis and autoradiography One-dimensional gel electrophoresis (I-DE) was performed according to Laemmli (1970). The acrylamide concentration was 10% in the separation gel and 3 0 in the stacking gel. The size of the gels was 130 x 140 x 0.75 ram. Two-dimensional gel electrophoresis (2-DE) was performed exactly as described by Jungblut and Seifert (1990). Briefly, the first dimension was a nonequilibrium pH gel electrophoresis (NEPHGE) in 4% rod gels (75 x 1.5 mm) containing 9 M urea. The cathode solution consisted o f 9 M urea, 5% glycerol and 5% ethylenediamine, the anode solution of 3 M urea and 742 mM phosphoric acid, Protein samples of 150 #g were applied at the anodic end of the gels. Isoelectric focusing was performed resulting in i841 Vhs. For the second dimension the focused rod gels were laid on 15% slab gels (Laemmli, 1970), using a Mini Protean Ii cell (Bio-Rad). The 1- and 2-DE gels were stained with Coomassie Brilliant Blue and dried. Radioactively labeled proteins were detected by autoradiography using Kodak X-Omat A R 5 X-ray film with intensifying screen. RESULTS

Photolytic labeling with [ot--~eP]GTP N u c l e a r a n d p l a s m a m e m b r a n e s were a n a l ~ d f o r G T P - b i n d i n g p r o t e i n s using p h o t o l y t i c labeling w i t h

411

GTP-binding proteins in nuclear membranes [ct-32p]GTP. In both subcellular fractions several proteins could be labeled as indicated by multiple bands in the autoradiogram (Fig. 1). Concerning the nuclear membranes, there was heavy labeling of proteins with a M , of 60 to 64 kDa, which, however, also occurred in the absence of u.v. irradiation. Besides this there was prominent labeling of proteins of 54, 51, 46, 39 and 29 kDa. In samples which contained 5 #M nonradioactive GTP the labeling of all proteins was strongly suppressed. With plasma membrane strong labeling in the region between 45 and 66 kDa could be observed. Additionally, the labeling of proteins with a Mw of 39, 37, 34 and 21 kDa could be seen, whereas no radioactivity could be detected at 29 kDa. Again, most of the labeling could be suppressed by nonradioactive GTP, indicating specific labeling.

A DP ribosylation with pertussis toxin As we could detect proteins of 39 kDa which could be photolytically labeled with [a-32p]GTP not only in plasma but also in nuclear membranes, we asked if these proteins are members of the group of heterotrimeric G-proteins. G-proteins can be quite specifically demonstrated by ADP ribosylation by bacterial toxins. Pertussis toxin, especially, very seleco

MW 205

tively modifies members of the Gi/o-family. Figure 2 shows the result of an A D P ribosylation experiment, where prior to the ADP ribosylation proteins were photolabeled with nonradioactive GTP. We found that in such double labeling experiments the signals were larger than in A D P ribosylation experiments without that prelabeling. A D P ribosylation of nuclear membrane proteins leads to the selective labeling of a protein of 39 kDa. This also holds for the plasma membrane, but in this case the labeling is much more intense. To quantify the ratio of labeling, we cut the respective bands out of the dried gel after autoradiography and determined the radioactivity by scintillation counting. We found that the radioactivity associated with the protein from nuclear membranes was 4.5% of the activity associated with the protein from plasma membrane. This figure exactly corresponds to the 4.5% contamination of nuclei with plasma membranes as revealed by determination of the specific activity of 5'nucleotidase in both fractions. o

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Fig. 1. GTP-binding proteins of nuclear and plasma membranes. Proteins were photolyticaUy labeled with [=-?2P]GTP, separated on SDS-PAGE (10%) and autoradiographed. (a) Nuclear membranes. Lanes: 1, without u.v. irradiation; 2, with u.v. irradiation; 3, as 2 in the presence of 5/iM GTP. Co)Plasma membranes. Lanes: 4, without

u.v. irradiation ; 5, with u.v. irradiation ; 6, as 5 in the presence of 5/~M GTP.

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Fig. 2. ADP ribosylation with pertussis toxin. Proteins of nuclear (a) and plasma membranes (b) were first subjected to photolytic labeling by nonradioactive GTP and then ADP ribosylated by pertussis toxin. Autoradiography after SDSPAGE (10%).

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For a detailed analysis of the question of whether the 29 kDa GTP-binding protein in nuclear membranes is unique to them or also present in other subcellular fractions, we used photolabeling in con> bination with 2-DE. Figure 3(b) shows the 2-DE pattern of photolabeled GTP-binding proteins in nuclear membranes. Besides the heavily labeled proteins in the region of 46 and 56 kDa again the 29 kDa protein already found in the t-DE cquld be detected. It apparently has a rather acidic pI of