- Email: [email protected]
Multiple ubiquitin conjugates are present in rat brain synaptic membranes and postsynaptic densities
ELSEVIER
Neuroscience Letters 168 (1994) 238 242
NIUROSCIINC[ lETTERS
Multiple ubiquitin conjugates are present in rat brain synaptic membranes and postsynaptic densities A.P. C h a p m a n a, S.J. S m i t h b, C.C. R i d e r ", P.W. B e e s l e y ~* "Department oJ'Biochemisto,, Royal Holloway and Bed/brd New College, University o/London, Egham. Surrey T[120 OEX, UK ~'Department qf Neurology, SmithKline Beecham, Harbin; UK Received 26 July 1993: Revised version received 19 December 1993: Accepted 21 December 1993
Abstract
The pattern of ubiquitin-protein conjugates present in a range of adult rat forebrain subcellular fractions has been investigated by immunoblotting with a monoclonal antibody specific for ubiquitin and its conjugates. Each fraction contains a complex and characteristic pattern of ubiquitin conjugates. Many integral synaptic membrane proteins are ubiquitinated, including a subset of high M r (> 120 kD) concanavalin A-binding glycoproteins. Postsynaptic densities are also enriched in ubiquitin conjugates, the profile being distinct from that of synaptic membranes. These results suggest that many plasma membrane and synaptic proteins are ubiquitinated. Key words: Ubiquitin: Glycoprotein: Postsynaptic density: Synaptic membrane; Plasma membrane; Monoclonal antibody
Ubiquitin is a highly conserved, 76 amino acid polypeptide, thought to be present in all eukaryote cell types. It is commonly found covalently conjugated to a diverse range of proteins. A well established role for this ubiquitination is to target the conjugated protein for rapid, non-lysosomal degradation (reviewed in ref. 1). However, the observation that some ubiquitinated proteins, notably histone H2A, are long-lived [2], suggests that ubiquitin has other functional roles. Because both free ubiquitin and its associated enzymic pathways are cytoplasmic, the ubiquitination of plasma membrane proteins has received comparatively little attention. However, ubiquitination of cell surface proteins could be of regulatory importance, for instance in the turnover and down-regulation of receptors and adhesion molecules. Indeed there is evidence for the ubiquitination of plasma membrane proteins including the lymphocyte homing receptor L-selectin (LECAM-1, Mel-14 antigen) (reviewed in ref. 3), rabbit growth hormone receptor [4], the 65 kD tumour necrosis factor receptor from HL60 cells [5], and murine platelet-derived growth factor recep-
*Corresponding author. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(93)E0889-4
tor [6]. Polyubiquitination of the latter receptor i's induced by ligand occupancy and is implicated in ligandinduced down-regulation of the receptor [7]. Limited evidence suggests that ubiquitin may also regulate neuronal cell surface function. Incubation of intact rat synaptosomes with anti-ubiquitin antiserum results in inhibition of several high-affinity neurotransmitter uptake systems, including those of glutamate and choline [8]. This suggests that a component of the uptake systems is ubiquitinated at a surface accessible site. Recently the presence of multiple ubiquitin conjugates has been demonstrated in subcellular fractions, including synaptic membrane (SM) and microsomes, prepared from hippocampi of control and ischemic rats [9]. Ubiquitin is also a component of the pathological inclusion bodies characteristic of several neurodegenerative disorders including Alzheimer's and Parkinson's disease [10]. In the present work we have studied the distribution of ubiquitin-protein conjugates in subcellutar fractions prepared from adult rat forebrain. We establish the presence of populations of these conjugates in the synapsederived synaptic membrane (SM) and postsynaptic density (PSD) fractions, and as integral components, some glycosylated, of the plasma membrane.
239
A. 1~ Chapman el aL / Neuroscience Letters 16~ I' 1994) 238 2 4 2
Subcellular fractions including SMs were prepared from adult Wistar rat forebrains using well established methods [11,12]. PSDs were prepared by the phase-partitioning m e t h o d o f G u r d et al. [13]. The protein, glycoprotein and electron-microscope (SM and PSDs only) profiles agree with the published data from other laboratories [11 13]. All samples were prepared in the presence o f a cocktail o f protease inhibitors consisting o f 0.2 m M P M S F and 1/ag/ml each o f leupeptin, pepstatin, chymostatin and antipain. Concanavalin A (Con A) binding glycoproteins were isolated from SMs by lectin affinity c h r o m a t o g r a p h y using C o n A agarose columns [14]. Endoglycosidase F/N-glycosidase F ( E n d o F) digestions were carried out according to the suppliers" instructions (Boehringer), at p H 8, optimal for the N-glycosidase F activity. Samples were separated by electrophoresis on sodium dodecyl sulphate (SDS) polyacrylamide gels (10% w/v) and Western blotted as previously described [15], using a wet blotting system (Bio-Rad). Blots were i m m u n o d e veloped with a murine anti-ubiquitin m o n o c l o n a l antib o d y ( M A b ) raised against SDS-denatured bovine ubiquitin-keyhole limpet h e a m o c y a n i n ( K L H ) conjugate. The SDS-denatured i m m u n o g e n raises rabbit antisera which preferentially bind ubiquitin conjugates c o m p a r e d to free ubiquitin [16]. In E L I S A our M a b reacts strongly with commercial preparations o f bovine and yeast ubiquitins, as well as the u b i q u i t i n - K L H conjugate, but fails to react with unconjugated K L H . I m m u n o r e a c t i v i t y on Western blots was routinely visualised using a peroxidase-conjugated secondary antibody, followed by the E C L detection system (Amersham). Blots o f brain homogenates developed in the absence o f the M a b were entirely negative. A Western blot o f adult rat forebrain subcellular fractions, i m m u n o d e v e l o p e d with our M A b , is shown in
1
L]
2
.
-180, -116 - 84 -58 -48
Mi
C
My LM SM Mt
1801168458483727-
liB-- . w ~ "
I
Fig. 1. Western blot of subcellular fractions from adult rat forebrain. Samples of homogenate (H), microsomes IMi), cytosol (C), myelin (My), light membranes (LM), synaptic membranes ISM) and mitochondria (Mt), each loaded at 45 rug protein, were Western blotted and immunodexeloped with the anti-ubiquitin MAb. Prominent SM-entithed bands are labelled with arrowheads, and in descending order arc of M~ 105, 72, 60, 41 and 38 kD, respectively. Free ubiquitm tUB) is also labelled. The positions and M~ in kD of standard markers are indicated to the left of the blot. Fig. 1. Each o f the fractions exhibits a characteristic and complex pattern o f immunoreactivity. As expected, only the cytosol is enriched with a b a n d corresponding to free ubiquitin. Intense high M.. immunoreactivity is detected in the h o m o g e n a t e and microsomal fraction, while other fractions such as myelin and m i t o c h o n d r i a contain relatively low immunoreactivity. SM contains multiple immunoreactive bands (for Mr see legend), together with diffuse high Mr staining. Some immunoreactive bands (see arrowheads) such as the species o f Mr 105, 72, 60, 41 and 38 k D are enriched in microsomes and SM. Some o f the SM bands are not detected in the less immunoreac-
4
3
~
H
5
6
7
I
-37-27 -
Fig. 2. lmmunoblot of SM extracted with alkaline carbonate (A) and Triton X-114 (B). A: samples of SM (lane 1), residual pellet (lane 2) and solubilised material (lane 3) were each loaded at 45/gg. Prominent bands in the residual pellet material, labelled with arrowheads, and are of Mr 156, 140, 93 and 72 kD, respectively. B: samples of SM (lane 4), residual insoluble material (lane 5), detergent phase (lane 6) and aqueous phase fractions (lane 7) were each loaded at 45/~g. Prominent bands in the residual insoluble material are labelled with arrowheads, and are of M. 156, 93 and 72 kD, and in the aqueous phase fraction, prominent bands of Mr 140 (ripper arrowhead) and 47 kD (lower arrowhead) are labelled. The positions and M~ in kD of standard markers are indicated between the blots.
240
.4.P (_Ttapman el ~d./ r¥~.'lgl*f).]('lf.'~'~t,'[" l.ctters 16,~' ~1'V94, 23,~ 242
1
2
pleted by rapid hydrolysis without further conjugation on ATP depletion. In order to exclude the possibility that SM ubiquitin protein conjugates were loosely bound extrinsic proteins, or had artefactually associated with the membranes during the fractionation procedure, SMs were extracted with 0.1 M sodium carbonate, pH 11.3 [17]. This procedure resulted in solubilisation of 40% of the SM protein. An
A
3727-
2
3
2
3
180-~i 1164-
Fig. 3. Ubiquitin conjugates in PSDs. Samples of SM (lane l ) and PSDs (lane 2) were each loaded at 45/2g. Prominent bands in the PSDs are labelled with arrowheads, and are of Mr 156, 72 and 47 kD. Positions and M r in kD of standard markers are indicated to the left of the blot.
58-~ 483727-
tive LM, which contains plasma membranes predominantly of non-synaptic origin. Comparison of the Western blot with silver stained gels (not shown) reveals that the MAb detects only a selected population of the protein species present. Immunodevelopment of Western blots with affinity purified rabbit anti-ubiquitin antiserum, raised by us as described [16], gives a virtually identical pattern to that shown in Fig. 1 (not shown). Samples prepared in the absence of the protease inhibitor cocktail show considerable loss of immunoreactivity commensurate with cleavage of the conjugates by endogenous ubiquitin hydrolase. When cultured cerebellar granule cells were incubated for 30 rain in the presence of 1/2g/ml oligomycin and 10 mM 2-deoxyglucose immediately prior to addition of SDS sample buffer, the immunoreactivity of all bands seen in the untreated samples was markedly reduced, although no change in the protein profile was detectable in silver stained gels (data not shown). This marked loss of immunoreactivity demonstates that the Mab is highly specific for conjugated ubiquitin, the conjugates being de-
g
1
18011168458483727-
C Fig. 4. Immunoblot of Con A-binding and Endo F digested material. A: Western blot of SM (lane 1, 45/xg), Con A-binding material isolated from 450/lg SM (lane 2) and non-Con A-binding material (lane 3, 45 ktg). Prominent immunoreactive species in the Con A-binding fraction are labelled with arrowheads, and are of M e 156, 140 and 122 kD. B: Western blot identical to A, but developed with biotinylated Con A and Vectastain biotinylated, avidin cross-linked peroxidase using chloronaphthol as substrate. C: Western blot of Con A-binding material from 450/2g SM (lanes 1 and 2) and non-Con A-binding material (lanes 3 and 4, 45/sg). Lanes 2 and 4 were digested with Endo F, and lanes 1 and 3 are corresponding control samples. An arrowhead marks the band of 112 kD in the non-Con A-binding fraction that is not detected after Endo F digestion. The positions and Mr in kD of the standard markers are indicated between the blots.
1
3727-
1
2
3
4
£
,4.P Chapman et aL / Neuroscience Letters 16,~' ( lC)C~4J 23,~' 242
immunoblot of the resulting fractions (Fig. 2A) shows that compared to the control SM (lane 1), the majority of the immunoreactivity remains bound to the residual membrane pellet (lane 2), and includes prominent bands of M, 156, 140, 93 and 72 kD. Only weak, predominantly high Mr immunoreactivity is detected in the solubilised fraction. This result suggests that the majority of SM ubiquitinated species are tightly associated with the membrane. The hydrophobicity of SM ubiquitin-protein conjugates was investigated by phase-partitioning with Triton X-114 [18]. The extraction results in a near equal distribution of protein into the detergent phase (37%), the aqueous phase (30%) and the residual insoluble material (33%). Each of these fractions exhibit markedly different patterns of immunoreactive polypeptides (Fig. 2B). The detergent phase material (lane 6) contains only weak immunoreactivity compared to the other two fractions. The aqueous phase (lane 7) is more intensel) immunoreactive, and several prominent species are observed including those of M, 140 and 47 kD. Intense immunoreactivity is also detected in the residual insoluble material (lane 5), and species present include those of M~ 156, 93 and 72 kD. The presence of a substantial proportion of SM ubiquitin-protein conjugates in the Triton X-114 insoluble fraction suggests that some of these species may be associated with the detergent insoluble postsynaptic densities (PSDs) present in this fraction. PSDs prepared from adult rat forebrain were indeed found to be rich in ubiquitin protein conjugates IFig. 3, lane 2). The pattern of immunoreactivity is, however, different from the parent SM, and some bands appear to be enriched in the PSDs. Several prominent immunoreactive bands can be detected in this fraction, including those of M,. 156, 72 and 47 kD. Since most if not all cell-surface proteins are glycosylated, and SMs are enriched in high mannose glycoproteins [19], we have investigated the presence of ubiquitinprotein conjugates in an SM Con A-binding fraction. The results show the presence of several prominent high M~ ubiquitin-protein conjugates in this fraction (Fig. 4A, lane 2), including those of M,. 156, 140 and 122 kD. Weaker bands of M.. 101 and 87 kD are also seen. In addition to these discrete bands, diffuse high M~ immunoreactivity is also observed. Only a subset of Con A-binding glycoproteins are ubiquitinated since development of Western blots of this material with biotinylated Con A (Fig. 4B, lane 2) shows that most lower Mr (< 120 kD) glycoprotein bands are not ubiquitinated. Many of the immunoreactive polypeptides in the non-binding fraction (Fig. 4A, lane 3) correspond to those detected in the parent SM (Fig. 4A, lane 1), but are clearly different from the immunoreactive Con A-binding glycoproteins (Fig. 4A, lane 2). The high loading of Con A binding material employed (lane 2) indicates that only a small
241
proportion of ubiquitin-protein conjugates bind to Con A. In order to confirm that the Con A-binding ubiquitinprotein conjugates are glycosylated, Endo F digestions were carried out. This enzyme mixture hydrolyses Nlinked carbohydrates [20] which account for 93% of oligosaccharides attached to SM glycoproteins [21]. A marked change in the pattern of ubiquitin-protein conjugates is observed following Endo F digestion of the Con A-binding material, consistent with a reduction in M. of the majority of these bands (Fig. 4C, lanes 1 and 2). In contrast, Endo F digestion of non-Con A-binding material as expected, has little effect (lanes 3 and 4), with the migration of only one conjugate, Mr 1 12 kD, being altered. It is likely that this ubiquitin protein-conjugate contains only non-Con A-binding, complex-type oligosaccharides. In this study, we have demonstrated that many integral membrane proteins of the rat forebrain are ubiquitinated. The observations that some ubiquitin-protein conjugates are present in SM but not LM, and that many are associated with the substantially pure PSD fraction suggests that they may be synaptic proteins. We have also demonstrated that SMs contain several high M, ubiquitinated glycoproteins, though their identity at present is unknown. Overall, our results show that in addition to the reported data on degenerative disorders, many proteins present in healthy' brain tissue are ubiquitinated. A priority is now to establish the function of this ubiquitination in specific subcellular compartments. APC holds a SERC-CASE studentship supported by SmithKline Beecham. [1] Hershko, A. and Ciechanover, A., The ubJqultin system l\~r prorein degradation, Annu. Rex'. Biochem., 61 (1992) 761 807. [2] Wu, R.S., Kohm K.W. and Bonnet, W.M., Metabolism of ubiquitinaled histones, J. Biol. Chem., 256 119811 5916 5920, [3] Siegehnan, M. and Weissman, I.L., Lymphocyte homing receptors, ubiquitin and cell surface proteins. In M. Rechstemer (Ed.), Ubiquitin, Plenuln, New York. 1988, pp. 239 269. [4] Leung, D.W., Spencer, S.A.. ('achmnca, G., Hammonds, R.G., Collins, C.. Henzel. W.J.. Barnard, R.. Waters, M,J. and Wood, W.I., Growth hormone receptor and serum binding protein: purification, cloning and expression, Nature, 330 11987) 537 543. [5] Loetschcr, H., Schlaeger, kl., Lahm, H.-W., Pan, Y.-('.E., Lesslauer, W. and Brockhaus, M., Purification and partial amino acid sequence analys~sof two distinct turnout necrosis factor receptors from HL6(Icells, J. Biol. ('hem., 265 (1990) 20131 2(1138. [6] Yarden, Y.. Escobedo, J.A., Kuang, W.-J.. Yang-Feng, T.L., Daniel, T.O., Tremble, P.M.. Chen, E.Y., Ando, M.E., Harkins, R.N., Francke, kl., Fried, V.A., Ullrich, A. and Williams, L.T.. Structure of the receptor for platelet-derived growth factor helps dcfine a family of closely'related growth factor receptors, Nature, 323 (19861 226 232. [7] Morl, S., Heldin. C.-H. and Claesson-Welsh, k., Ligand-induced polyubiquitination of the platelet-derived growth factor fl-receptor, J. Biol. Chem.. 267 119921 6429 6434. [8] Meyer, E.M., West, C.M.. Stevens, B.R., Chau, V., Nguyen, M.-T. and Judkins, J.H., Ubiquitin-directed antibodies inhibit neuronal transporters m rat brain synaptosomcs, J. Neurochem., 49 (1987) 1815 1819.
242
A . P Chapman et al /Neuroscience Letters 16~' t 1994 J 23~ 242
[9] Hayashk T., Takada, K. and Matsuda, M., Subcellular distribution of ubiquitin-protein conjugates in the hippocampus following transient ischemia, J. Neurosci. Res., 31 (1992) 561 -564. [10] Lowe, J., Blanchard, A., Morrell, K., Lennox, G., Reynolds, L., Billett, M., Landon, M. and Mayer, R.J., Ubiquitin is a common factor in intermediate filament bodies of diverse type in man, including those of Parkinson's disease, Pick's disease and Alzheimer's disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease, J. Pathol., 155 (1988~ 9~ 15. [11] Cotman, C.W. and Taylor, D., Isolation and structural studies on synaptic complexes from rat brain, J. Cell Biol., 55 (1972) 696-711. [12] DeSilva, N.S., Gurd, J.W. and Schwartz, C., Developmental alteration of rat brain synaptic membranes. Reaction of glycoproteins with plant lectins, Brain Res., 165 11979) 283 293. [13] Gurd, J.W., Gordon-Weeks, R and Evans, W.H., Biochemical and morphological comparison of postsynaptic densities prepared from rat, hamster and monkey brains by phase partitioning, J. Neurocbem., 39 (1982) 1117--1124. [14] Beesley, RW., Paladino, T., Gravel, C., Hawkes, R. and Gurd, J.W., Characterisation ofgp50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody, Brain Res.. 408 (1987) 65 78.
115] Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic Lransli.er of proteins t¥om polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc. Natl. Acad. Sci. USA, 7in 11979) 4350 4354. [16] Hershko, A., Eytam E., Ciechanover. A. and ttaas, A.L.. lmmunochemical analysis of the turnover of intraccllular ubiquitin protein conjugates in intact ceils. Relationship t{) the breakdown of abnormal proteins. J. Biol. Chem., 257 (1982} 13964-11370. [17] Howell, K.E. and Palade, G.E., Hepatic Golgi fractions resolved into membrane and content subfractions, .l. t,cll Biol., 92 (19821 822 832. [18] Bordier, C., Phase separation of integral membrane proteins ill Triton X-114 solution, J. Biol. Chem., 256 (19811 1604- 1607. [19] Gurd, J.W., Glycoproteins of the synapse. In R.U. Margolis and R.K. Margolis (Eds.), Neurobiology of the Glycoconjugates, PlellUln, New York, 1989, pp. 219 242. [20] Elder. J.H. and Alexander, S., Endo-,B-N-acet~lglucosaminidase:endoglycosidase from Havohacterium meningosepticum that cleaves both high-mannose and complex glycoproteins, Proc. Natl. Acad. Sci. USA, 79 (19821 4540-4544. [21] Krusius, T., Finne, J., Margolis, R.U. and Margolis, R.K., Structural features of microsomal, synaptosomal, mitochondrial, and soluble glycoproteins of brain. Biochemistry, 17 (1078) 3849 3854.
Neuroscience Letters 168 (1994) 238 242
NIUROSCIINC[ lETTERS
Multiple ubiquitin conjugates are present in rat brain synaptic membranes and postsynaptic densities A.P. C h a p m a n a, S.J. S m i t h b, C.C. R i d e r ", P.W. B e e s l e y ~* "Department oJ'Biochemisto,, Royal Holloway and Bed/brd New College, University o/London, Egham. Surrey T[120 OEX, UK ~'Department qf Neurology, SmithKline Beecham, Harbin; UK Received 26 July 1993: Revised version received 19 December 1993: Accepted 21 December 1993
Abstract
The pattern of ubiquitin-protein conjugates present in a range of adult rat forebrain subcellular fractions has been investigated by immunoblotting with a monoclonal antibody specific for ubiquitin and its conjugates. Each fraction contains a complex and characteristic pattern of ubiquitin conjugates. Many integral synaptic membrane proteins are ubiquitinated, including a subset of high M r (> 120 kD) concanavalin A-binding glycoproteins. Postsynaptic densities are also enriched in ubiquitin conjugates, the profile being distinct from that of synaptic membranes. These results suggest that many plasma membrane and synaptic proteins are ubiquitinated. Key words: Ubiquitin: Glycoprotein: Postsynaptic density: Synaptic membrane; Plasma membrane; Monoclonal antibody
Ubiquitin is a highly conserved, 76 amino acid polypeptide, thought to be present in all eukaryote cell types. It is commonly found covalently conjugated to a diverse range of proteins. A well established role for this ubiquitination is to target the conjugated protein for rapid, non-lysosomal degradation (reviewed in ref. 1). However, the observation that some ubiquitinated proteins, notably histone H2A, are long-lived [2], suggests that ubiquitin has other functional roles. Because both free ubiquitin and its associated enzymic pathways are cytoplasmic, the ubiquitination of plasma membrane proteins has received comparatively little attention. However, ubiquitination of cell surface proteins could be of regulatory importance, for instance in the turnover and down-regulation of receptors and adhesion molecules. Indeed there is evidence for the ubiquitination of plasma membrane proteins including the lymphocyte homing receptor L-selectin (LECAM-1, Mel-14 antigen) (reviewed in ref. 3), rabbit growth hormone receptor [4], the 65 kD tumour necrosis factor receptor from HL60 cells [5], and murine platelet-derived growth factor recep-
*Corresponding author. 0304-3940/94/$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(93)E0889-4
tor [6]. Polyubiquitination of the latter receptor i's induced by ligand occupancy and is implicated in ligandinduced down-regulation of the receptor [7]. Limited evidence suggests that ubiquitin may also regulate neuronal cell surface function. Incubation of intact rat synaptosomes with anti-ubiquitin antiserum results in inhibition of several high-affinity neurotransmitter uptake systems, including those of glutamate and choline [8]. This suggests that a component of the uptake systems is ubiquitinated at a surface accessible site. Recently the presence of multiple ubiquitin conjugates has been demonstrated in subcellular fractions, including synaptic membrane (SM) and microsomes, prepared from hippocampi of control and ischemic rats [9]. Ubiquitin is also a component of the pathological inclusion bodies characteristic of several neurodegenerative disorders including Alzheimer's and Parkinson's disease [10]. In the present work we have studied the distribution of ubiquitin-protein conjugates in subcellutar fractions prepared from adult rat forebrain. We establish the presence of populations of these conjugates in the synapsederived synaptic membrane (SM) and postsynaptic density (PSD) fractions, and as integral components, some glycosylated, of the plasma membrane.
239
A. 1~ Chapman el aL / Neuroscience Letters 16~ I' 1994) 238 2 4 2
Subcellular fractions including SMs were prepared from adult Wistar rat forebrains using well established methods [11,12]. PSDs were prepared by the phase-partitioning m e t h o d o f G u r d et al. [13]. The protein, glycoprotein and electron-microscope (SM and PSDs only) profiles agree with the published data from other laboratories [11 13]. All samples were prepared in the presence o f a cocktail o f protease inhibitors consisting o f 0.2 m M P M S F and 1/ag/ml each o f leupeptin, pepstatin, chymostatin and antipain. Concanavalin A (Con A) binding glycoproteins were isolated from SMs by lectin affinity c h r o m a t o g r a p h y using C o n A agarose columns [14]. Endoglycosidase F/N-glycosidase F ( E n d o F) digestions were carried out according to the suppliers" instructions (Boehringer), at p H 8, optimal for the N-glycosidase F activity. Samples were separated by electrophoresis on sodium dodecyl sulphate (SDS) polyacrylamide gels (10% w/v) and Western blotted as previously described [15], using a wet blotting system (Bio-Rad). Blots were i m m u n o d e veloped with a murine anti-ubiquitin m o n o c l o n a l antib o d y ( M A b ) raised against SDS-denatured bovine ubiquitin-keyhole limpet h e a m o c y a n i n ( K L H ) conjugate. The SDS-denatured i m m u n o g e n raises rabbit antisera which preferentially bind ubiquitin conjugates c o m p a r e d to free ubiquitin [16]. In E L I S A our M a b reacts strongly with commercial preparations o f bovine and yeast ubiquitins, as well as the u b i q u i t i n - K L H conjugate, but fails to react with unconjugated K L H . I m m u n o r e a c t i v i t y on Western blots was routinely visualised using a peroxidase-conjugated secondary antibody, followed by the E C L detection system (Amersham). Blots o f brain homogenates developed in the absence o f the M a b were entirely negative. A Western blot o f adult rat forebrain subcellular fractions, i m m u n o d e v e l o p e d with our M A b , is shown in
1
L]
2
.
-180, -116 - 84 -58 -48
Mi
C
My LM SM Mt
1801168458483727-
liB-- . w ~ "
I
Fig. 1. Western blot of subcellular fractions from adult rat forebrain. Samples of homogenate (H), microsomes IMi), cytosol (C), myelin (My), light membranes (LM), synaptic membranes ISM) and mitochondria (Mt), each loaded at 45 rug protein, were Western blotted and immunodexeloped with the anti-ubiquitin MAb. Prominent SM-entithed bands are labelled with arrowheads, and in descending order arc of M~ 105, 72, 60, 41 and 38 kD, respectively. Free ubiquitm tUB) is also labelled. The positions and M~ in kD of standard markers are indicated to the left of the blot. Fig. 1. Each o f the fractions exhibits a characteristic and complex pattern o f immunoreactivity. As expected, only the cytosol is enriched with a b a n d corresponding to free ubiquitin. Intense high M.. immunoreactivity is detected in the h o m o g e n a t e and microsomal fraction, while other fractions such as myelin and m i t o c h o n d r i a contain relatively low immunoreactivity. SM contains multiple immunoreactive bands (for Mr see legend), together with diffuse high Mr staining. Some immunoreactive bands (see arrowheads) such as the species o f Mr 105, 72, 60, 41 and 38 k D are enriched in microsomes and SM. Some o f the SM bands are not detected in the less immunoreac-
4
3
~
H
5
6
7
I
-37-27 -
Fig. 2. lmmunoblot of SM extracted with alkaline carbonate (A) and Triton X-114 (B). A: samples of SM (lane 1), residual pellet (lane 2) and solubilised material (lane 3) were each loaded at 45/gg. Prominent bands in the residual pellet material, labelled with arrowheads, and are of Mr 156, 140, 93 and 72 kD, respectively. B: samples of SM (lane 4), residual insoluble material (lane 5), detergent phase (lane 6) and aqueous phase fractions (lane 7) were each loaded at 45/~g. Prominent bands in the residual insoluble material are labelled with arrowheads, and are of M. 156, 93 and 72 kD, and in the aqueous phase fraction, prominent bands of Mr 140 (ripper arrowhead) and 47 kD (lower arrowhead) are labelled. The positions and M~ in kD of standard markers are indicated between the blots.
240
.4.P (_Ttapman el ~d./ r¥~.'lgl*f).]('lf.'~'~t,'[" l.ctters 16,~' ~1'V94, 23,~ 242
1
2
pleted by rapid hydrolysis without further conjugation on ATP depletion. In order to exclude the possibility that SM ubiquitin protein conjugates were loosely bound extrinsic proteins, or had artefactually associated with the membranes during the fractionation procedure, SMs were extracted with 0.1 M sodium carbonate, pH 11.3 [17]. This procedure resulted in solubilisation of 40% of the SM protein. An
A
3727-
2
3
2
3
180-~i 1164-
Fig. 3. Ubiquitin conjugates in PSDs. Samples of SM (lane l ) and PSDs (lane 2) were each loaded at 45/2g. Prominent bands in the PSDs are labelled with arrowheads, and are of Mr 156, 72 and 47 kD. Positions and M r in kD of standard markers are indicated to the left of the blot.
58-~ 483727-
tive LM, which contains plasma membranes predominantly of non-synaptic origin. Comparison of the Western blot with silver stained gels (not shown) reveals that the MAb detects only a selected population of the protein species present. Immunodevelopment of Western blots with affinity purified rabbit anti-ubiquitin antiserum, raised by us as described [16], gives a virtually identical pattern to that shown in Fig. 1 (not shown). Samples prepared in the absence of the protease inhibitor cocktail show considerable loss of immunoreactivity commensurate with cleavage of the conjugates by endogenous ubiquitin hydrolase. When cultured cerebellar granule cells were incubated for 30 rain in the presence of 1/2g/ml oligomycin and 10 mM 2-deoxyglucose immediately prior to addition of SDS sample buffer, the immunoreactivity of all bands seen in the untreated samples was markedly reduced, although no change in the protein profile was detectable in silver stained gels (data not shown). This marked loss of immunoreactivity demonstates that the Mab is highly specific for conjugated ubiquitin, the conjugates being de-
g
1
18011168458483727-
C Fig. 4. Immunoblot of Con A-binding and Endo F digested material. A: Western blot of SM (lane 1, 45/xg), Con A-binding material isolated from 450/lg SM (lane 2) and non-Con A-binding material (lane 3, 45 ktg). Prominent immunoreactive species in the Con A-binding fraction are labelled with arrowheads, and are of M e 156, 140 and 122 kD. B: Western blot identical to A, but developed with biotinylated Con A and Vectastain biotinylated, avidin cross-linked peroxidase using chloronaphthol as substrate. C: Western blot of Con A-binding material from 450/2g SM (lanes 1 and 2) and non-Con A-binding material (lanes 3 and 4, 45/sg). Lanes 2 and 4 were digested with Endo F, and lanes 1 and 3 are corresponding control samples. An arrowhead marks the band of 112 kD in the non-Con A-binding fraction that is not detected after Endo F digestion. The positions and Mr in kD of the standard markers are indicated between the blots.
1
3727-
1
2
3
4
£
,4.P Chapman et aL / Neuroscience Letters 16,~' ( lC)C~4J 23,~' 242
immunoblot of the resulting fractions (Fig. 2A) shows that compared to the control SM (lane 1), the majority of the immunoreactivity remains bound to the residual membrane pellet (lane 2), and includes prominent bands of M, 156, 140, 93 and 72 kD. Only weak, predominantly high Mr immunoreactivity is detected in the solubilised fraction. This result suggests that the majority of SM ubiquitinated species are tightly associated with the membrane. The hydrophobicity of SM ubiquitin-protein conjugates was investigated by phase-partitioning with Triton X-114 [18]. The extraction results in a near equal distribution of protein into the detergent phase (37%), the aqueous phase (30%) and the residual insoluble material (33%). Each of these fractions exhibit markedly different patterns of immunoreactive polypeptides (Fig. 2B). The detergent phase material (lane 6) contains only weak immunoreactivity compared to the other two fractions. The aqueous phase (lane 7) is more intensel) immunoreactive, and several prominent species are observed including those of M, 140 and 47 kD. Intense immunoreactivity is also detected in the residual insoluble material (lane 5), and species present include those of M~ 156, 93 and 72 kD. The presence of a substantial proportion of SM ubiquitin-protein conjugates in the Triton X-114 insoluble fraction suggests that some of these species may be associated with the detergent insoluble postsynaptic densities (PSDs) present in this fraction. PSDs prepared from adult rat forebrain were indeed found to be rich in ubiquitin protein conjugates IFig. 3, lane 2). The pattern of immunoreactivity is, however, different from the parent SM, and some bands appear to be enriched in the PSDs. Several prominent immunoreactive bands can be detected in this fraction, including those of M,. 156, 72 and 47 kD. Since most if not all cell-surface proteins are glycosylated, and SMs are enriched in high mannose glycoproteins [19], we have investigated the presence of ubiquitinprotein conjugates in an SM Con A-binding fraction. The results show the presence of several prominent high M~ ubiquitin-protein conjugates in this fraction (Fig. 4A, lane 2), including those of M,. 156, 140 and 122 kD. Weaker bands of M.. 101 and 87 kD are also seen. In addition to these discrete bands, diffuse high M~ immunoreactivity is also observed. Only a subset of Con A-binding glycoproteins are ubiquitinated since development of Western blots of this material with biotinylated Con A (Fig. 4B, lane 2) shows that most lower Mr (< 120 kD) glycoprotein bands are not ubiquitinated. Many of the immunoreactive polypeptides in the non-binding fraction (Fig. 4A, lane 3) correspond to those detected in the parent SM (Fig. 4A, lane 1), but are clearly different from the immunoreactive Con A-binding glycoproteins (Fig. 4A, lane 2). The high loading of Con A binding material employed (lane 2) indicates that only a small
241
proportion of ubiquitin-protein conjugates bind to Con A. In order to confirm that the Con A-binding ubiquitinprotein conjugates are glycosylated, Endo F digestions were carried out. This enzyme mixture hydrolyses Nlinked carbohydrates [20] which account for 93% of oligosaccharides attached to SM glycoproteins [21]. A marked change in the pattern of ubiquitin-protein conjugates is observed following Endo F digestion of the Con A-binding material, consistent with a reduction in M. of the majority of these bands (Fig. 4C, lanes 1 and 2). In contrast, Endo F digestion of non-Con A-binding material as expected, has little effect (lanes 3 and 4), with the migration of only one conjugate, Mr 1 12 kD, being altered. It is likely that this ubiquitin protein-conjugate contains only non-Con A-binding, complex-type oligosaccharides. In this study, we have demonstrated that many integral membrane proteins of the rat forebrain are ubiquitinated. The observations that some ubiquitin-protein conjugates are present in SM but not LM, and that many are associated with the substantially pure PSD fraction suggests that they may be synaptic proteins. We have also demonstrated that SMs contain several high M, ubiquitinated glycoproteins, though their identity at present is unknown. Overall, our results show that in addition to the reported data on degenerative disorders, many proteins present in healthy' brain tissue are ubiquitinated. A priority is now to establish the function of this ubiquitination in specific subcellular compartments. APC holds a SERC-CASE studentship supported by SmithKline Beecham. [1] Hershko, A. and Ciechanover, A., The ubJqultin system l\~r prorein degradation, Annu. Rex'. Biochem., 61 (1992) 761 807. [2] Wu, R.S., Kohm K.W. and Bonnet, W.M., Metabolism of ubiquitinaled histones, J. Biol. Chem., 256 119811 5916 5920, [3] Siegehnan, M. and Weissman, I.L., Lymphocyte homing receptors, ubiquitin and cell surface proteins. In M. Rechstemer (Ed.), Ubiquitin, Plenuln, New York. 1988, pp. 239 269. [4] Leung, D.W., Spencer, S.A.. ('achmnca, G., Hammonds, R.G., Collins, C.. Henzel. W.J.. Barnard, R.. Waters, M,J. and Wood, W.I., Growth hormone receptor and serum binding protein: purification, cloning and expression, Nature, 330 11987) 537 543. [5] Loetschcr, H., Schlaeger, kl., Lahm, H.-W., Pan, Y.-('.E., Lesslauer, W. and Brockhaus, M., Purification and partial amino acid sequence analys~sof two distinct turnout necrosis factor receptors from HL6(Icells, J. Biol. ('hem., 265 (1990) 20131 2(1138. [6] Yarden, Y.. Escobedo, J.A., Kuang, W.-J.. Yang-Feng, T.L., Daniel, T.O., Tremble, P.M.. Chen, E.Y., Ando, M.E., Harkins, R.N., Francke, kl., Fried, V.A., Ullrich, A. and Williams, L.T.. Structure of the receptor for platelet-derived growth factor helps dcfine a family of closely'related growth factor receptors, Nature, 323 (19861 226 232. [7] Morl, S., Heldin. C.-H. and Claesson-Welsh, k., Ligand-induced polyubiquitination of the platelet-derived growth factor fl-receptor, J. Biol. Chem.. 267 119921 6429 6434. [8] Meyer, E.M., West, C.M.. Stevens, B.R., Chau, V., Nguyen, M.-T. and Judkins, J.H., Ubiquitin-directed antibodies inhibit neuronal transporters m rat brain synaptosomcs, J. Neurochem., 49 (1987) 1815 1819.
242
A . P Chapman et al /Neuroscience Letters 16~' t 1994 J 23~ 242
[9] Hayashk T., Takada, K. and Matsuda, M., Subcellular distribution of ubiquitin-protein conjugates in the hippocampus following transient ischemia, J. Neurosci. Res., 31 (1992) 561 -564. [10] Lowe, J., Blanchard, A., Morrell, K., Lennox, G., Reynolds, L., Billett, M., Landon, M. and Mayer, R.J., Ubiquitin is a common factor in intermediate filament bodies of diverse type in man, including those of Parkinson's disease, Pick's disease and Alzheimer's disease, as well as Rosenthal fibres in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease, J. Pathol., 155 (1988~ 9~ 15. [11] Cotman, C.W. and Taylor, D., Isolation and structural studies on synaptic complexes from rat brain, J. Cell Biol., 55 (1972) 696-711. [12] DeSilva, N.S., Gurd, J.W. and Schwartz, C., Developmental alteration of rat brain synaptic membranes. Reaction of glycoproteins with plant lectins, Brain Res., 165 11979) 283 293. [13] Gurd, J.W., Gordon-Weeks, R and Evans, W.H., Biochemical and morphological comparison of postsynaptic densities prepared from rat, hamster and monkey brains by phase partitioning, J. Neurocbem., 39 (1982) 1117--1124. [14] Beesley, RW., Paladino, T., Gravel, C., Hawkes, R. and Gurd, J.W., Characterisation ofgp50, a major glycoprotein present in rat brain synaptic membranes, with a monoclonal antibody, Brain Res.. 408 (1987) 65 78.
115] Towbin, H., Staehelin, T. and Gordon, J., Electrophoretic Lransli.er of proteins t¥om polyacrylamide gels to nitrocellulose sheets: Procedure and some applications, Proc. Natl. Acad. Sci. USA, 7in 11979) 4350 4354. [16] Hershko, A., Eytam E., Ciechanover. A. and ttaas, A.L.. lmmunochemical analysis of the turnover of intraccllular ubiquitin protein conjugates in intact ceils. Relationship t{) the breakdown of abnormal proteins. J. Biol. Chem., 257 (1982} 13964-11370. [17] Howell, K.E. and Palade, G.E., Hepatic Golgi fractions resolved into membrane and content subfractions, .l. t,cll Biol., 92 (19821 822 832. [18] Bordier, C., Phase separation of integral membrane proteins ill Triton X-114 solution, J. Biol. Chem., 256 (19811 1604- 1607. [19] Gurd, J.W., Glycoproteins of the synapse. In R.U. Margolis and R.K. Margolis (Eds.), Neurobiology of the Glycoconjugates, PlellUln, New York, 1989, pp. 219 242. [20] Elder. J.H. and Alexander, S., Endo-,B-N-acet~lglucosaminidase:endoglycosidase from Havohacterium meningosepticum that cleaves both high-mannose and complex glycoproteins, Proc. Natl. Acad. Sci. USA, 79 (19821 4540-4544. [21] Krusius, T., Finne, J., Margolis, R.U. and Margolis, R.K., Structural features of microsomal, synaptosomal, mitochondrial, and soluble glycoproteins of brain. Biochemistry, 17 (1078) 3849 3854.