Ubiquitin in health and disease

Biochimica et Biophysico Acta, 1089 (1991) 141-157 © 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 016747819100139W

141

Review

BBAEXP 92262

Ubiquitin in health and disease R. John May~.r, Jane Arnold, Lajos L~szl6, Michael Landon and James Lowe Departments of Biochemistry and i Pathology, Unit~rsity of Nottingham Medical School, Queens Medical Centre, Nottingham (U.K) (Received 6 March 1991)

Key words: Ubiquitin; Ubiquitin-mediated proteolysis; Neurodegenerative disease; Viral disease; Development

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Perceived functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Serendipity: happy and unexpected discoveries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. New vistas: diagnosis, molecular pathogenesis and cell biological roles . . . . . . . . . . . . . . . . .

141 142 142 142

I!.

Ubiquitin and chronic neurodegenerative disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Commonality in the major human and animal chronic neurodegenerative disorders . . . . . . . . B. Significance for neuropathological diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mechanisms of neurodegeneration: role of ubiquitin and the cell stress response . . . . . . . . . .

143 143 146 146

I11.

Ubiquitin and the lysosome system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. A neuropathological hint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Biochemical and immunogold electron microscopical findings . . . . . . . . . . . . . . . . . . . . . . .

147 147 147

IV.

Ubiquitin: a unifying cofactor in non-lysosomal and lysosomal protein catabolism? . . . . . . . . . . . A. A common covalent signal for protein degradation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Extralysosomal protein degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

149 149 151

V.

Ubiquitin in virally infected cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

151

VI.

Ubiquitin and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

153

VII. Ubiquitin cross-reactive protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

154

VIII. Evolutionary perspective on ubiquitin and the eukaryotic cell . . . . . . . . . . . . . . . . . . . . . . . . . .

154

IX.

155

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

155

1. I n t r o d u c t i o n It is n o t i n t e n d e d t h a t t h i s r e v i e w s h o u l d b e a c o m p r e h e n s i v e s u r v e y o f all t h e b i o c h e m i c a l s t u d i e s o n

Correspondence: R.J. Mayer, Departments of Biochemistry and Pathology, University of Nottingham Medical School, Queens Medical Centle, Nottingham NG7 2UH, U.K.

ubiquitin: instead, we present a personalised description of the emergence of ubiquitin as a central player in t h e fields o f m o l e c u l a r p a t h o l o g y a n d m o l e c u l a r cell biology. T h e r e a r e s e v e r a l e x c e l l e n t r e v i e w s o n t h e current understanding of the biochemistry of ubiquitin t o w h i c h t h e r e a d e r is r e c o m m e n d e d [ i - 5 ] . T h i s s h o r t p r e a m b l e s h o u l d o b v i a t e t h e n e e d f o r t h e a l m o s t oblig a t o r y b e g i n n i n g o f u b i q u i t i n r e v i e w s w h i c h is: ' U b i q u i t i n is a s m a l l b a s i c p r o t e i n o f 76 a m i n o a c : ~ s

142 which is one of the most evolutionary conserved proteins currently known' - or did it!

1-,4. Perceived functions It is interesting, with the benefit of hindsight, to reflect on whether progress in the understanding of the functions of ubiquitin would have taken a different path if most of the initial elegant work [6-8] had been done on a cell type which retains its nucleus, rather than on the reticulocyte, which is in the process of becoming an anucleate erythrocyte. The extensive intracellular reorganisation of the reticuiocyte during maturation involves the need to eliminate and degrade unwanted proteins and organelles. This requirement probably explains the substantial activity of the ubiquitin related enzyme system [9] and subserving p r o teinases in the maturing reticuiocyte which facilitated the important discovery that the covalent coupling of ubiquitin to target proteins is a signal for non-lysosomal proteolysis [6,7]. The maturing reticulocyte loses much more tha~ the nucleus and mitochondria, discarding all other intracellular membranous organelles as well. Could some functions of ubiquitin be thrown away in the maturing reticulocyte with the elimination of these membranebound organelles? In other nucleated cell types certain plasma membrane receptors are ubiquitinated [10,11]. Whatever for, if protein ubiquitination is confined to cytosolic proteins destined for non.lysosomal degradation? The non-cytosolic functions of ubiquitin continue to remain enigmatic. It has been known for many years that some of the nuclear histones are ubiquitinated, although the reason for the conjugation of ubiquitin to histories H2A and H2B is not fully understood [12,13]. It has been suggested that protein ubiquitination may be related to gene expression [14]. However, protein ubiquitination is clearly related to other nuclear functions. Two of the ubiquitin conjug~z~ng enzymes (Ee's), CDC 34 and RAD 6 in yeast are involved in cell cycle control and radiation repair, respectively [15,16,117]. Protein ubiquitination, therefore has several roles in the nucleus, it is interesting from an evolutionary viewpoint to consider if some of the nuclear functions of ubiquitin were modified and adapted for roles outside the nucleus with the development of increasingly complex cytoplasmic functions (section VIII). l B . Serendipity

Much scientific progress has been based on the single-minded preoccupation of workers with just one model .system. It is interesting and curious that this philosophy can be at the expense of further progress. For instance, over many years of study on a protein

cofactor for non-lysosomal protein catabolism, it never occurred to anyone to ask whether ubiquitin had a role in protein elimination in disease. However, serendipity came along and showed that ubiquitin does indeed play a role in some of the most intractable human and animal neurodegenerative diseases as well as in other chronic degenerative and viral diseases. The exact role of protein ubiquitination in the course of disease progression is still unknown, but the molecular pathological observations have thrown up a whole new set of questions which have indicated new functions for ubiquitin in the extensive architectural changes that occur in diseased cells.

I.C. New vistas The application of basic biochemical findings to the study of clinical problems has at least four aspects. The primary survey of the distribution of a molecule in diseased t!ssues, in this case by the application of ubiquitin immunocytochemistry, can serve to identify an unsuspected commonality in disorders which were previoasly seen as unrelated in mechanistic terms [18]. This has served to bring together a group of ostensibly disparate chronic degenerative diseases into a family of conditions characterised by some form of intracellular ubiquitinated inclusion. The second aspect is that the application of sensitive analytical techniques for the detection of novel molecules can lead to new diagnostic approaches. Again this is true for ubiquitin immunocytochemistry that has become obligatory for the unambiguous diagnosis of certain chronic neurode. generative diseases [29]. There is a more difficult, and yet in the long term most beneficial, third aspect of the application of basic biochemical findings to clinical problems which is its value for understanding of molecular pathogenesis and disease progression. Intracellular ubiquitin-protein deposits have again certainly provided new insights into several degenerative and viral diseases and raised new questions about the cellular capacity to respond to stress, damage and injury. One fundamental evolutionary question is whether a life process based on the single nucleated cell could ever have advanced in the early stages without the development of genes for intracellular protein products which could recognise order and disorder in the cell, eliminate problem proteins and organelles and generally distinguish self and non-self cytoplasm. Such a question then begs others which include: what are the functions of such genes in a multicellular organism and what role do such genes have in diseased cells? Lastly, how do the intracellular products of such genes interact with what has come to be known as the immune system? A final benefit of the interplay between pathology and basic biomedical science is the opportunity to make molecular pathological observations which

143

spawn new experimentation that gives rise to findings which widen the perception of the basic functions of a molecule in cellular physiology. A classical case was the discovery of microsomes whilst searching for Rous sarcoma virus [17]. The discovery of the enrichment of ubiquitin-protein conjugates in what could be lysosomes in hippocampal neurones in AIzheimer's disease [18] with the subsequent finding of ubiquitin-protein conjugates in lysosomes in a variety of cell types may be yet another example of the value of close cooperation between molecular cell biology and pathology. The combined basic scientific and molecular pathological

studies on ubiquitin have provided data which suggest that ubiquitin may have a role in both non-lysosomal and lysosomal proteolysis in eukaryotic cells. 11. Ubiquitin and chronic neurodegenerative disease

II-A. Commonality in the major human and animal chronic neurodegeneratit'e disorders The first indication that ubiquitin would have significant clinical impact came from studies on neurodegenerative diseases. Within a few months several iaborato-

B

,r\ •

P (

#

o

C

Q

~ ~'~

Fig,I. ]mmunohistochemical demonstration of ubiquitin-protein conjugates in fiJamentoos inclusions in chronic degenerat~'e ~scases. (A) CoMical neurofibrilla~ tangle in Alzh©imer's disease. (B) Conical l.~wy body in diffuse Lewy body disease. (C) Hepat~-yte Mallory hody in alcoholic liver disease. (D) Filamentous inclusiOn in anterior horn neurone in motor neurone disease.

144 ries reported that ubiquitin is a component of neurofibrillary tangles (Fig. IA) in Alzheimer's disease [19-21]. The discovery in lhara's laboratory [19] is noteworthy since it came about by identifying ubiquitin as the antigen for a monoclonal antibody with affinity for paired helical filaments. The molecular characterisation of the other protein components of neurofibrillary tangles in Alzheimer's disease is still incomplete; the tangles contain the microtubule associated protein tau [22], although there is considerable debate as to whether neurofilaments are also present. It was against this contentious background that some serendipitous 'happy and unexpected discoveries' took place. The most sensitive element of the cell cytoskeleton to cell stress (heat-shock) is the intermediate filament system [23]. Studies designed to understand the degradative fate of Sendal virus transmembranous ~lycoproteins transplanted into the membrane of cells in culture had shown that rapid vimentin intermediate filament collapse is coincident with the propulsion of the glycoproteins to a perinuclear location where the proteins enter the autophagolysosome system [24]. The intermediate filaments subsequently rapidly extend again. The reversible filament changes are complete in a few hours whereas the viral glycoproteins are degraded slowly (tl/2 50-100 h) in the lysosomal system. These observations could have remained on the library shelves gathering dust were it not for the findings on neurofibrillary tangles! The basic curiosities were: if ubiquitin is associated with one type of filamentous inclusion in a neurodegenerative disease, could it be a component of other distinct filamentous inclusions ,.',1 other neurodegenerative diseases? If ubiquitin was found to be associated with such inclusions then would any of them contain intermediate filaments? The formation of collapsed filamentous inclusions, composed, at least in part, of intermediate filaments containing ubiquitinated proteins, might indicate some connection between intermediate filaments, ubiquitin, the lysosomai system and protein catabolism. Much of this surmise turns out to be correct, the remainder requires further experimentation! The neuropathological detection of the neuronal inclusions called Lewy bodies in the brain stem in Parkinson's disease is pathognomonic for the disease. Brain stem Lewy bodies have been shown to contain neurofilaments, one of the intermediate filament classes. Brain stem Lewy bodies contain ubiquitin-protein deposits [18,25-27]. Another form of Lewy body had been reported in the cortical regions of the brain as a very rare cause of dementia called diffuse Lewy body disease [28]. The application of ubiquitin immunocytochemistry has revealed two unexpected important features: namely, that cortical Lewy bodies also contain ubiquitin-protein deposits (Fig. 1B ), and also that diffuse Lewy body disease is not rare bat is a

common cause of dementia accounting for up to one third of cases [29]. The antiserum used to show that ubiquitin is present in these inclusions detects the carboxyl-terminus of the molecule and shows a marked preference for ubiquitin-protein conjugates: ubiquitin chemically conjugated to a carrier protein completely blocks the immunoreactivity of inclusions, whereas free ubiquitin only marginally suppresses immunoreactivity [18]. Furthermore, Western blots show the presence of ubiquitin-protein conjugates in some of the intracellular inclusions (e.g., paired helical filaments in new rofibrillary tangles [19] and Mallory bodies in alcoholic liver disease; R.J. Mayer and J. Lowe, unpublished). On these grounds the inclusions are considered to contain primarily ubiquitin-protein conjugates rather than free ubiquitin. Some cerebellar astrocytomas contain filamentous inclusions called Rosenthal fibres which are aggregates of gliai filament acidic protein-containing filaments. ~[.,ese inclusions also possess ubiquitin-protein conjugates [30]. Thus, a second type of inclusion containing intermediate filaments is associated with protein ubiquitination. There is a third class of intermediate filament-containing inclusion associated with ubiquitinated proteins, the cytokeratin-based Mallory body (Fig. IC), which is found in hepatocytes in alcoholic liver disease. Clearly, therefore, the formation of inclusions characterised by the presence of ubiquitinated proteins is not confined to the brain. Finally, there is a rare cytoplasmic body myopathy in which the desmincontaining filamentous inclusions can also be demonstrated to have ubiquitinated proteins [18]. in short, some intracellular filamentous inclusions which exhibit, at least in part, intermediate filament epitopes, also contain ubiquitin-protein conjugates. The mechanism of formation of these inclusions is currently unknown, as is the role of ubiquitin in inclusion body biogenesis. Ultrastructural analysis will reveal much more about these inclusions. Ubiquitin immunocytochemistry has also revealed unsuspected filamentous inclusions in another neurological disease. Spinal cord anterior horn neurones [31,32] and motor neurones in the brain cortex [33] can be seen to contain ubiquitinated filamentous inclusions (e.g., Fig. 1D) in amyotrophic lateral sclerosis (motor neurone disease). This filamentous inclusion had not been detected before the advent of ubiquitin immunocytochemistry. The filaments often seem to pervade and surround a region of the neurone previously seen histologically to contain an area of rarefaction called a Bunina body [34]. In motor neurone disease there is a large increase (two-fold) in the expression of the polyubiquitin C gene in the surviving spinal anterior horn neurones and in the brain [35]. The polyubiquitin C gene has a heat-shock promoter and codes for nine intronless repeats in man. The expression of the poly-

145 ubiquitin B gene which codes for three ubiquitins is also probably elevated [35]. The combined increase in the expression of the polyubiquitin genes is presumably required to satisfy a greater need for ubiquitin in the degenerating neurones. It will be interesting to discover whether there are comparable changes in the expression of polyubiquitin genes in the other ncurodegenerative diseases in the family of disorders with intraceilular ubiquitin-protein deposits. From the preceding paragraphs it is apparent that ubiquitin immunocytochemistry has identified an unsuspected commonality in some neurodegenerative diseases, including the major human chronic neurological disorders (Alzheimer's disease. Parkinson's disease, diffuse Lewy body disease and amyotrophic lateral sclerosis). The co~,.ditions are all part of a family of ubiquitin-filament diseases. However, filamentous inclusions containing ubiquitin-protein deposits are not invariably found in neurological disorders. Thus, multiple sclerosis and the consequences of some spinal injuries can be c:~;:~,,!ed from the family of diseases [32]. This observatit,..~, must be significant for our understanding of the reasons for the active use of ubiquitin in inclusion body biogenesis. It suggests that some common molecular response occurs in chronically degenerating neurones which involves ubiquitin and other cell-stress (heat-shock) proteins. This presence of ubiquitin-protein conjugates in intraneuronal filamentous inclusions has served to identify a commonality in the major human neurodegeneratire diseases which presumably indicates shared molecular mechanisms in the neuronal response to disease stimulus and progression. Recently, there have been immunohistochemical observations which serve to separate the diseases, indicating either distinct routes to the formation of ubiquitinated filamentous inclusions or different stages in inclusion biogenesis in the various diseases. Ubiquitin is not only conjugated to proteins but can also be removed. There is a family of enzymes involved in the deconjugation of ubiquitinated proteins [36]. It has been recently shown that one of these ubiquitin carboxyl-terminal hydrolases [37], an abundant brain protein previously called PGP 9.5, is present in Lewy bodies in diffuse Lewy body disease and in Rosenthal fibres in cerebeilar astrocytomas but is only found occasionally in globose neurofibrillary tangles in Alzheimer's disease. The hydrolase is not seen in the filamentous inclusions of amyotrophic lateral sclerosis but afflicted neurones do seem to exhibit considerably elevated cytoplasmic levels of the enzyme. The removal of ubiquitin from some target proteins seems to be occurring in some intracellular inclusions in brain but not in others; this presumably reflects differences in ubiquitin-related metabolic activity in the various types of inclusion. The activation of a generalised cell-stress response

in degenerating neurones in certain of the diseases is indicated by another recent finding, that the protein a B crystallin is found immunohistochcmically in Lewy bodies and Rosenthal fibres but again not in neurofibrillary tangles [39]. The a B crystallin gene has a heatshock promoter which may well be activated in some degenerating neurones. Although the aB crystallin is homologous in the carboxyl-terminal region with the small heat-shock proteins, the antiserum used in these studies was raised by the synthetic peptide route and only detects the 20 kDa subonit of aB crystallin on Western blots in extracts of both human and animal tissues known to be enriched in a B crystallin [39]. The intracellular role of a B crystallin is not understood, but its presence can be observed in a variety of normal [38,39] as well as diseased tissues, e.g., tumours in man [39]. The protein appears to be expressed at high levels in cells subjected to oxidative stress [38] and those involved in protein uptake or secretion, e.g., thyroid follicular cells and colonic epithelium, respectively [39]. Clearly, the molecular characteristics of all of the intrancuronai filamentous inclusions are not identical. Thus far, the most signif'w.anl findings for the neuroticgenerative diseases are that two molecular markers, the ubiquitin carboxyl-terminai hydrolase and a B cry~ tallin, are not routinely found in neurofibrillary tangles in Alzheimer's disease or the filamentous inclusions in amyotrophic lateral sclerosis. The proteins are predominantly confined to inclusions (Lewy bodies, Rosenthal fibres) which unequivocally contain intermediate fdaments. These observations are further supported by the finding of a B crystallin in the cytokeratin-containing Mallory bodies in alcoholic liver disease [39]. Hypotheses relating to the role of the heatshock gene products in inclusion body biogenesis will be discussed later, but it is already clear that further studies on other enzymes and proteins involved in protein ubiquitination are required in order to understand fully the cell stress response and inclusion body formation in neurodegenerative diseases. The presence of filamentous inclusions containing ubiquitin-protein deposits is not confined to the diseases indicated above; they are also found in brains from other chronic neurodegenerative disorders, including Pick's disease [18,27], Down's syndrome [40,~11] and dementia pugilistica [40]. Nor are ubiquitinated filamentous inclusions confined to the cytoplasm of neurones; they have been observed in Mariuesco bodies seen in the nucleus of neurones in patients with intranuclear hyaline inclusion disease [42]. Ultrastructuraily these inclusions are filamentous and it is tempting to speculate that this type of inclusion may involve nuclear lamins, yet another member of the family of intermediate filaments [43]. In the neurodegenerative diseases ubiquitinated proteins are not only associated with filamentous inclu-

146 sions in the cell bodies of neurones. In AIzheimer's disease and diffuse Lewy body disease ubiquitin-protein deposits are found in distended nearites surrounding extracellular senile plaques and throughout the neuropil (the brain regions between the neuronal cell bodies and containing the numerous nerve processes). In Alzheimer's disease the immunostaining of ubiquitin conjugates in the distended neurites of plaques is not unlike that seen for lysosomal cathepsins [44]. There are also ubiquitin-protein conjugates in areas of granuio-vacuolar degeneration in hippocampal neurones in Alzheimer's disease [18]. The immunostaining of ubiquitin deposits in distended neurites and in granulovacuoles is not restricted to human idiopathic neurodegenerative diseases but is also seen in a transmissible neuropathy in a mouse model of scrapie [45]. The neurites surrounding the extracellular prion plaques show immunostaining for ubiquitinated proteins and some neurones also contain ubiquitin-protein deposits in what appear to correspond to the giant autophagic vacuoles previously described in a mouse model of Creutzfcldt-Jakob disease [46]. Ubiquitin immunoreactivity is also seen in human brain in cases of Creutzfeldt-Jakob disease, kuru [47] and Gerstmann-Straussler-Scheinker syndrome [116]. There are, therefore, parallels between the molecular pathologies of the transmissible animal and human encephalopathies and the idiopathic human neurodegenerative diseases. Protein ubiquitination is clearly activated in chronic diseases which result in neurodegeneration and serves to unify the molecular pathology of these disorders.

liB. Significance for neuropathological diagnosis Ubiquitin immunocytochemistry has certainly changed, if not revolutionised, the neuropathological diagnosis of neurodegenerative disease and has become a 'hallmark' [48] for the neuropathological diagnosis of diffuse Lewy body disease and amyotrophic lateral sclerosis [31,32]. Ubiquitin immunocytochemistry has revealed the full significance of cortical Lewy body disease which, by conventional histopathological techniques, had previously been largely overlooked. Immunocytology with antisera to ubiquitin-protein conjugates is the most sensitive method for the detection of cortical Lewy bodies [48]. Studies on the East coast [49] and West coast [50] of the United States have confirmed those in Europe [48,51] which show that diffuse Lewy body disease is, after Alzheimer's, disease which covers about half of the cases, the second most common cause of dementia accounting for a further third. More investigations are needed, but it does appear that Alzheimer's disease is characterised by intraneutonal neurofibrillary tangles and extracellular senile

plaques, while diffuse Lewy body disease has intraneuronal cortical Lewy bodies with similar senile plaques. Other differences between the diseases include the presence of abnormally phosphorylated tau proteins only in the neurofibrillary tangles and dystrophic neurites of the plaques in Alzheimer's disease, and, in contrast, the previously mentioned presence of a ubiquitin carboxyl-terminal hydrolase and a B crystallin only in the cortical Lewy bodies in diffuse Lewy body disease.

II-C. Mechanisms of neurodegeneration: role of ubiquitin and the cell stress response The existing molecular neuropathological findings suggest two ostensibly opposite and apparently conflicting explanations for the involvement of ubiquitin in neurodegenerative disease. Protein ubiquitination may cause neuronal death, e.g., as part of a pathologicallyactivated irreversible catabolic process. Alternatively, protein ubiquitination may be part of a cytoprotective process trying to rescue neurones from the chronic continuous onslaught of damaged neuronal proteins or organelles. There is, of course, a separate proposal, namely that protein ubiquitination may be initially cytoprotective in a neurone and, if the process cannot combat the neuronal degenerative insult, then ubiquitin-dependent protein catabolism is involved in destroying neurones in the final stages of nerve cell death. There is experimental evidence (section VI) that, in the special case of hormonally-programmed neuromuscular cell death after eclosion in an insect, polyubiquitin gene expression is considerably elevated to produce the ubiquitin necessary for the extensive destruction of cellular material [98,99]. Also, during early embryonic development (section Vl) it is apparent [100] that protein ubiquitination is involved in the extensive remodelling which occurs in cells undergoing the major reorganisation which accompanies differentiation. These findings are not surprising when it is remembered that ubiquitin is involved in catabolic processes which can be activated for different reasons to subserve alternative physiological needs. The necessity for changes in polynbiquitin gene expression to meet the need for ubiquitin for these processes is more understandable when seen against the putative role of ubiquitin in both non-lysosomal and lysosomal protein degradation (section liD. The cytoprotective role of protein ubiquitination probably occurs in neurones at preterminal stages of disease progression in neurodegeneration [52,53] There are several reasons for thinking that this should be the case. First, of all the cell types in muiticellular organisms it might be expected in neurones that genes involved in combatting cell stress and injury should be most sensitive to activation. Death of nerves is rapidly

147 followed by the death of the organism cf. the effects of nerve gases. The notion nf an exquisitely sensitive cell stress response in the central nervous system is supported by studies on stroke, where it has been shown in models that ubiquitin and heat-shock protein 70 (hsp 70) expression is an immediate response to hypoxia in hippocampal neurones [54,55]. Cell survival appears to directly correlate with the degree of activation of ubiquitin gene expression. Secondly, in the adult very few nerve cells are replaced and therefore there must be effective ways to combat injurious stimuli and to eliminate the affected molecules when protein or organelle damage occurs. Such a response would naturally involve the cell stress (heat-shock) genes. Some of the cell-stress gene products are increasingly seen to be involved as 'molecular chaperones' in organelle biogenesis, e.g., hsp 70, hsp 60 [111,112]. The problems of organeile biogenesis in an ordinary cell are compounded in nerves by the vastly greater distances over which organeiles may have to travel by orthograde flow. It would not be surprising, therefore that heatshock proteins should be used in some aspect of these processes and also be required in supranormai amounts in diseased neurones. Finally, the possibility that protein ubiquitination is involved in molecular chaperoning in a biosynthetic sense should not be overlooked. It has been shown that antiserum raised against ubiquitin can block the biosynthetic insertion of an enzyme into the outer mitochondrial membrane of the mitochondrion [56]. Ubiquitin could be used in some similar way to form the ubiquitinated inclusions in cells as well as for some protein catabolic functions. Understanding the role of ubiquitin in neurodegenerative diseases would be made much easier if the purpose of inclusion body formation was understood; a problem that is made more difficult to resolve by the fact, already discussed, that there are at least two classes of filamentous inclusion bodies both in terms of molecular characteristics and, possibly also, mode of biogenesis. The inclusion bodies which clearly contain intermediate filaments may be viewed with more confidence since, as indicated previously, the inclusions share molecular features irrespective of whether the inclusions are within neurones or other types of cell: intermediate filaments are also the element of the cytoskeleton most sensitive to cell stress [23]. While it has been suggested that ubiquitinated filamentous inclusions form because of some downstream failure of the ubiquitin dependent non-lysosomal protein degradation system [19,20], it is also possible that the intermediate filament type of inclusion is an end point in itself and represents a cellular attempt to isolate and 'cocoon' unwanted cellular material within a filamentous structure. Such filamentously cocooned proteins or organelles may then become a permanent feature of an

injured cell or may be slowly eliminated by a ubiquitin coupled protein catabolic system. A further possibility is that all or part of an inclusion may be ejected from the cell [52,53,57]. A fuller picture of the role of heat shock proteins and intermediate filaments in the reaction to cell stress will be described in section V when cytomorphological rearrangments in virally infected cells are discussed.

111. Ubiquitin and the lysosomal system IliA. A neuropathological hint In some hippocampal neurones in Alzheimer's disease areas of granulovacuolar degeneration are found histologically. The granulovacuoles are membrane bound and ~ontain ubiquitin-protein conjugates [18]. The impossibility of obtaining appropriately fresh human brain tissue suitable for electron microscopy has up to now precluded the ultrastructural analysis of these granulovacuoles. However, there are at least two theoretical possibilities to explain their presence: the ubiquitinated material could either be present in some form of secretory vesicle or in autophagic vacuoles related to the lysosome system. There is no clear evidence for either option but the presence of large secretory vesicles in the cell bodies of hippocampal neurones appeared inherently unlikely. The possibility that ubiquitinated-protein conjugates might be enriched in the lysosome system of cells had not been considered.

III-R Biochemical and immunogold electron microscopical findings The notion that ubiquitinated proteins might accumulate in the lysosomes was initially tested by compromising the lysosomal system of fibroblasts with a potent inhibitor of thiol cathepsins called E-64 [58,59]. In these 'lysosomally-constipated' cells ubiquitinated-protein conjugates can be seen by immunohistochemistry to accumulate in lysosomes. Furthermore, ubiquitinprotein conjugates can be detected by Western blotting in subcellular fractions enriched in lysosomal marker enzymes. The combined studies indicate that ubiquitinated proteins can accumulate in the functionally compromised iysosomal system of fibroblasts. These observations are interesting but do not allow us to conclude that ubiquitinated proteins are directed to the lyso. some system of normal fibroblusts. While these studies were in progress there was a report based on immunogold electron microscopy which indicated that free ubiquitin is also found in the iysosomes of cells [60]. The distribution of ubiquitin-protein conjugates has been assessed in normal fibroblasts by immunogold electron microscopy and indicates that the gold patti-

I))

•

\ 4•

~" '-7 0

!

-~!

•

Fig. 2. lmmunogold electron microscopical analysis of the cellular distribution of ubiquitin-protein cor,jugates in different cell types. (A) in El~tein=Barr transformed lymphoblastoid cells. (D) Dense primary (azurophilic) granules in rabbit polymorphonuclear neutrophils. (Bat's = 0.2 ~m).

Ly~mes in 3T3-LI mouse fibroblasts. (B) Large a u t o p h a g o l ~ m e in E-64 treated 3T3-LI cells. IC) L ~ m e s

cles are selectively enriched some 12-fold in the lysosomes of these cells relative to all other organelles [61]. These findings suggest that ubiquitin=protein conjugates are normally enriched in the lysosomal system of fibroblasts. However, the nature of tSe ubiquitinated proteins is unknown. Immunoblotting experiments with

cell extracts from E-64 treated cells generally give immunoreactive smears of ubiquitin-protein conjugates on the nitrocellulose replicas [58,62]. The lysosomes may contain both intact and partially degraded ubiqui= tinated proteins and protein fragments [58,62] as well

as free ubiquitin [60] (Fig. 2).

149 Immunogold electron microscopy has also been used to show that ubiquitin-protein conjugates are enriched in the lysosome system of polymorphonuclear neutrophils [63] and Epstein-Barr virus transformed lymphoblastoid cells [64]. The latter is discussed in section V. The notion that ubiquitin-protein deposits in hippocampal neurones in AIzheimer's disease might be in autophagolysosomes has led to the experimental confirmation of the presence of ubiquitinated proteins in the lysosome system of fibroblasts, polymorphs and in iymphoblastoid cells; it has yet to be confirmed in neurones! The detection of some forms of ubiquitin-protein conjugates in lysosomes of cells was unexpected given the existing perceptions of the roles of ubiquitin in eukaryotic cells [2,65]. The novel findings concerning the functions of ubiquitin raise several questions, not least of which is: how do the conjugates enter the autophagolysomal system? There are several possibilities. The first is that protein ubiquitination may occur cotranslationally dragging one or more ubiquitin side chains, covalently attached to a nascent polypeptide, through the membrane as the polypeptide moves into the lumen of the endoplasmic reticulum. This proposal has already been put forward to explain the ubiquitination of the lymphocyte homing receptor [11]. The second possibility is that protein ubiquitination occurs in the endoplasmic reticulum and its topological equivalents; that is in essentially all membrane bound vesicles in the exocytotic and endocytotic pathways of the cell. These two alternative processes have not been tested out or reported experimentally. Such modes of ubiquitination would be expected to attach ubiquitin to many proteins destined for alternative organelles during their sorting in the secretory pathway. The ubiquitination of many proteins destined for different organelles would not lead to the specific enrichment of ubiquitin-protein conjugates in the lysosomal system of cells detected by immunogold electron microscopy [58,61,63,64]. Immunogoid electron microscopy of polymorphonuclear neutrophils emphasises the selcctivc prcsencc of the ubiquitin-protein conjugates in lysosome-related vesicles; the conjugates are present only in the primary (azurophilic) granules and not the secondary (specific) granules. The primary granules contain a complement of acid hydrolases, including proteinases, while the secondary granules enclose a variety of non-lysosomai anti-inflammatory proteins that are secreted from neutrophils in the inflammatory reaction, it is still not clear how the ubiquitinated proteins enter the primary azurophilic granules. It could occur during neutrophii maturation by an autophagic process or by heterophagy during the fusion of phagosomes with the primary granules in the inflammatory reaction. The latter possibility is unlikely since the studies were performed on

polymorphonuclear neutrophils isolated from bone marrow [63]. A third mechanism for the entry of ubiquitinated proteins into the lysosomal system is from the cytoplasm by a process of either microautophagy or macroautophagy. Immunogoid electron microscopy demonstrates clusters of gold particles at what appear to be microinvaginations at the surface of multivesicular bodies which are likely candidates for organelles undergoing microautophagy [58,61,64]. Cytoplasmic aggregates of proteins, including ubiquitinated proteins, may act as a trigger for the surface invagination necessary for microautophagy. Receptors for ubiquitinated proteins on the surface of such organelles would sense and control the microautophagic process. Recent studies on the lysosomal degradation of cellular proteins after cell stress have indicated that the protein ubiquitination system must be functionally intact for the process to occur [66]. In this way, studies on cells with temperature-sensitive mutations in the ubiquitin activating enzyme, E l, have shown that proteins which accumulate in stressed cells can only be degraded by the lysosome system at the permissive temperature. Protein ubiquitination appears to be necessary for the uptake of damaged or unwanted proteins into the iysosomes. The potent inhibitor of macroautophagy, 3-methyladenine, appears to block the uptake process. This has been interpreted to mean that protein ubiquitination is involved in promoting a process of macroautophagy in stressed cells. Macroautophagy involves the wrapping of a double membrane around an area of cytoplasm to form a membrane bound vacuole before fusion of the vacuole with a lysosome (Fig. 3B). It is not yet known how protein ubiquitination plays a role in such a complex process. The studies on cells with temperature sensitive mutations in the ubiquitin activating pathway [66] are also significant since they demonstrate the importance of temporal aspects of ubiquitination for iysosomai degradation of proteins. Other studies have demonstrated by immunogold electron microscopy the presence of ubiquitinprotein conjugates in the lysosome system of different cell types [58,61,63,64].

IV. Ubiquitin: a unifying cofactor in non-lysosomal and lysosomal protein catabolism [117]. IV-A. A common covalent signal for protein degradation ? Protein ubiquitination may have a variety of functions in eukaryotic cells including roles in chromatin structure, gene expression and repair, organelle biogenesis and protein catabolism [117]. Definitive evidence to prove all these suggestions is not yet forthcoming but much effort has been invested

150 A

S.E.R.

~.o

LF.

MI'II~NOND

Y/ C

Fig. 3. Possiblerolesof ubiquitinationin nuclearand lysosomefunction:(A-i) Suggestedstructural interactionsbetween intermediatefilaments in the nuclearlaminaand the nuclearenvelope.(A-ii)Role of protein ubiquitinationin the interactionsbetween intermediatefilamentsin the nuclear lamina and the nuclearenvelope.(B) Role of protein ubiquitinationin the interactionbetween intermediatefilamentsand smooth endoplasmic retieulum involvedin cytoplasmic sequestration during macroautophagy. (C) Role for protein ubiquitination in membrane invaginationof multivesicularbodies duringmicroautophagy,o.n.m.,outer nuclearmembrane;i.n.m.,innernuclearmembrane;S,E.R., smooth endoplasmic reticulum,I.F., intermediatefilament,M.V.B., multivesicularbody; Re, putative membranereceptor for ubiquitinatedprotein; Ub-X, ubiquitinatedprotein. in studying the role of ubiquitin in protein degradation. The current consensus is that protein ubiquitination is a signal for extra-lysosomal protein degradation (section IV-B). However, the recent findings on ubiquitinated proteins and the lysosomal system provide data for a unifying role of protein ubiquitination in directing proteins into either non-lysosomal or lysosomai pathways of protein catabolism. Once ubiquitinated a protein may have to run the gauntlet of either cytosolic proteinases or uptake into lysosomes.

The complexities of the addition of ubiquitin to proteins (section IV-B) [117] in terms of single or multiple ubiquitination and the lack of knowledge of effector molecules which recognise different types of ubiquitinated proteins preclude explanations of how a ubiquitinated protein becomes selected for non-lysosomal or lysoso~al protein degradation. The alternative notion that protein ubiquitination may be involved in driving membrane reorganisation (Fig. 3B) for macroautophagy [66] must also be considered. This

151 concept implies that all of the proteins destined for autophagy are not necessarily ubiquitinated. A role for ubiquitin in two major intraceilular proteolytic systems would indicate a previously unsuspected central function for ubiquitin as the primary covalent cofactor in intracellular protein catabolism. Furthermore, the concept would explain, at least in part, why ubiquitin is found only in eukaryotic cells, which have extensive endomembrane systems involved in autophagy and heterophagy. Some ideas on how ubiquitin could have evolved to assume such a role are considered in section VIII.

IV-B. Extralysosomal protein catabolic system The mechanisms involved in the selection of a protein for non-lysosomal degradation by the ubiquitin-dependent pathway have been clarified by integrating biochemical knowledge of the ubiquitin conjugating enzymes with model systems involving genetically engb neered protein substrates. In order for a protein to be degraded via the ubiquitin-dependent pathway at least two determinants appear to he necessary [67,68]. One is an N-terminal amino acid which is recognized by a uhiquitin protein ligase (E a) to which it reversibly binds according to an N-end rule [69]. The second is a 'mobile' lysine residue to which a branched multiubiquitin chain can be added: chain formation occurs by isopeptide linkage between the C-terminus of ubiquitin and lysine-48 of the preceding member of the chain. Such determinants can either be present on the same polypeptide chain or in the case of oligomeric proteins on different subunits [70]. It is this multiubiquitin chain that acts as the proteinase-recognition site for the specific ubiquitin-conjugate degrading enzyme [68]. Although it is still unclear how the muiti-ubiquitin chain is formed, a specific ubiquitin carrier protein has recently been identified that is able to ubiquitinate ubiquitin through lysine-48 and form multi-ubiquitin chains consisting of at least 10 and maybe as many as 47 ubiquitins [71]. This suggests that following binding of the substrate protein to E 3, a specific ubiquitin carrier protein is able to participate exclusively in multi-ubiquitin chain formation [117]. Although in vitro studies have demonstrated that multi-ubiquitination of substrate proteins targets them for degradation, there are only two cellular proteins of physiological importance that have been reported to undergo this reaction in vivo. One is the plant protein phytochrome, which following far-red light exposure, is converted to an unstable aggregated form which is subsequently multi-ubiquitinated and degraded. The aggregation of this chromoprotein into amorphous non-membrane bound granules activates ubiquitin-dependent catabolism [72]. More recently, cyclin, a pro-

tein whose removal is necessary for cells to progress into anaphase of the cell cycle, has been shown to be multi-ubiquitinated and degraded by a ubiquitin-dependent mechanism [73]. A highly conserved amino acid sequence in the N-terminal region of cyclin (designated as a so-called destruction box) has been identified as a signal for degradation. It is possible that a specific ubiquitin protein ligase, localized within the nucleus, recognizes this sequence [117]. Other examples of proteins which are appear to be degraded by the ubiquitin pathway include nuclear oncoproteins [74] and the a-globin chains in /3-thalassaemia [75]; the ubiquitin conjugate status of these proteins is, however, not known. It is expected that many more proteins will be shown to be multiubiquitinated and degraded by the ubiquitin-dependent pathway. There is, however, increasing evidence that populations of stable mono-ubiquitinated proteins exist in living cells. Ubiquitin has been shown to be present on a number of cell surface receptors [10,76-78] and in association with a form of actin [79]. The most common stable mono-ubiquitinated proteins are histones where 10% of intracellular ubiquitin is conjugated to H2A and H2B [80], through lysine-119 and 120, respectively [81,82]. From in vitro studies it appears that certain ubiquitin carrier proteins can multi-ubiquitinate histones by an E3-independent mechanism both by mono-ubiquitination of multiple lysine residues or by multi-ubiquitination of a single lysine residue [83-85]. Degradation of a multi-ubiquitinated histone (H3) by the ubiquitin-conjugate degrading proteinase has recently been described [86], suggesting that E3's are not essential for multi-ubiquitination and subsequent degradation of all target proteins. Nevertheless, although these in vitro studies are important in understanding the mechanisms and functions of the ubiquitin conjugating enzymes, the physiological relevance of the observations is yet to be proven. V. Ubiquitin in virally infected cells Given that ubiquitin may have several functions in eukaryotic cells it should not be surprising that ubiquitin has been found in different guises associated with animal and plant viruses. Ubiquitin-like sequences have been detected in the genomes of togavirus [87] and baculovirus [88]. In the latter case the gene is transcribed during the late phase of viral infection. The immediate-early protein (ICP-4) of herpes simplex virus specifically induces the human polyubiquitin B gene [89]. A tobacco mosaic virus coat protein can be ubiquitinated [90]. In a similar manner ubiquitin-protein conjugates are found in several plant virus particles [91] and ubiquitin is also associated with avian leukosis virus particles [92, 117].

152 The associations currently include: the possession of viral genomic sequences which may code for ubiquitinlike proteins; the induction of ubiquitin gene expression by viral infection; the detection of ubiquitinated proteins in viral particles; the enrichment of viral proteins in subcellular compartments containing ubiquitin-protein conjugates. Interpretations of the reasons for an association between ubiquitin and viral infection may be complicated by the many putative roles of ubiquitin in cell homeostasis. The finding of ubiquitin-like sequences in the nuclcic acids of viruses may indicate some chaperoning role of the ubiquitinlike molecule in viral replication. The induction of polyubiquitin gene expression and ubiquitination of viral proteins might also play a role in viral production. Alternatively, the induction of ubiquitin gene expression, formation of ubiquitin-viral protein conjugates and the detection of viral membrane proteins in lysosomes together with some form of ubiquitin-protein conjugate may indicate a cellular attempt to modify viral proteins in order to eliminate the proteins from the cell or prevent the normal oligomerisation of viral proteins into nucleoprotein particles. The association of the latent membrane protein of Epstein-Barr virus with ubiquitin-protein conjugates in lysosomes provides an example of where a process of viral protein elimination appears to be taking place in a viraily transformed cell [64]. Epstein-Barr virus transformed lymphoblastoid cells contain the majority of the intracellular latent membrane transforming protein of the virus in autophagolysosomes oriented with mitochondria around the microtubule organising centre. The lysosomes also contain an hsp 70, which is significant since a member of that protein family is involved in the uptake of a fraction of proteins having a KFERQ sequence motif into the lysosomes of cells subjected to the stress of serum deprivation [93]. The redistributed organelles appear to be entwined with vimentin-containing intermediate filaments. A variety of sizes of autolysosomes is seen in transformed cells; a minority of cells contain giant autophagolysosomes which may fuse with the cell surface to eject the contents of the giant vacuole. The transformed cells may therefore fragment [94], degrade to amino acids or eject the latent membrane protein from the cell [64]. lmmunocytochemistry shows that the filamentous inclusions in the Epstein-Barr transformed cells in part resemble those seen in the chronic human degenerative diseases (section If). Thus, the inclusions in Epstein-Barr transformed lymphoblastoid cells, previously visualised by immunofluorescence [95] as patches containing vimentin intermediate filaments as well as the viral latent membrane protein, are now seen in the most recen: studies also to contain ubiquitin-protein conjugates [64]. There are, therefore, some molecular

markers in common with certain of the inclusions in the human chronic degenerative diseases that contain both intermediate filaments and ubiquitin-protein conjugates (section II). The differences which have been observed are hsp 70 in the viral inclusions [64] contrasted with a B crystallin in the intermediate filament containing inclusions in the diseases [39]. Thus far the intermediate filament inclusions in the diseases have not been fully resolved at the ultrastrucrural level and the substructures of the inclusions and locations of the ubiquitin-protein conjugates remain unknown. In contrast, the intermediate filament inclusions in Epstein-Barr virus transformed lymphoblastoid cells have been resolved by immunogold electron microscopy to show that the viral membrane protein is in lysosome-related large vacuoles, which also contain ubiquitin-protein conjugates [64]. Since a variety of cell stresses can cause intermediate filament collapse (section II), it is attractive to suggest that in parallel with these observations the ubiquitin-protein conjugates may be similarly disposed in some autolysosome-like structure in the intermediate filament-containing inclusions of the chronic degenerative diseases. Such a proposal is reinforced by a fundamental similarity in the Epstein-Barr transformed ceils and the degenerating neurones of Alzheimer's disease, diffuse Lewy body disease and the transmissible encephalopathies; the similarity is that the cytomorphologicai rearrangements are all associated with the production and accumulation of a single membrane glycoprotein. In the Epstein-Barr transformed lymphoblastoid cell it is the latent membrane protein of the virus. It is remarkable that only a few of the numerous Epstein-Barr virus genes are expressed in transformed lymphoblastoid ceils. Thus, the few genes necessary for the episomal replication of the viral genome are expressed together with the gene for the latent membrane gly~.oprotein of the virus (there is recent evidence that there are two transcripts and proteins from the gene [96]). Alzheimer's disease and diffuse Lewy body disease are associated with the extracellular accumulation of a truncated peptide derived from a /3amyloid precursor membrane glycoprotein (reviewed in Ref. 97), while the transmissible encephalopathies are associated with the accumulation of a glypiated (glycosylphosphatidylinositol-anchored) membrane protein [113]. Very recently it has been shown that a point mutation in the/]-amyloid precursor protein results in a familial early-onset form of Alzheimer's disease [114] and transfection of a mutant prion gene corresponding to the form found in the human transmissible encephalopathy Gerstmann-Straussler-Scheinker syndrome (a familial form of Creutzfeldt-Jakob disease) results in rapid neurodegeneration with pathology resembling that seen in the human disease [115]. The

153 mutated proteins presumably present a difficult turnover problem to the neurones and cause a significantly more rapid disease. From the foregoing it seems appropriate to propose a general model in which the cor.tinuous production of a foreign or endogenous membrane glycoprotein is sufficient to trigger the reorganisation of the cell cytoskeleton. In those cases where intermediate filament inclusions occur, the accumulating glycoprotein or fragment of the protein may be found in some form of autophagolysosome associated with the inclusions. Finally, we must consider the possibility that the microarchitecturai rearrangements which occur in response to the continual stress of production and accumulation of a single membrane protein are a deliberate cell strategy designed to cocoon unwanted proteins for eventual elimination. In this way the formation of intermediate filament inclusions containing satellite auxiliary subeellular organelles, e.g., lysosomes and mitochondria, can be seen as the assembly of a novel (perhaps transitory) megaorganelle designed to isolate and eliminate deleterious proteins or damaged organelles from the cell.

VI. Ubiquitin and development Such a versatile posttranslational covalent protein modification as ubiquitination might be expected to be involved in some aspect of development or differentiation. The degradative functions of ubiquitination, particularly when widened to include both extraphagosoreal and lysosomal proteolysis could be utilised in different types of developmental situation, which could be as distinct as programmed cell death and the extensive intracellular reorganisational changes which occur in early embryos during morphogenesis. Recently, changes in polyubiquitin gene expression a n d / o r cytoplasmic protein ubiquitination have been shown to occur in these two types of developmental situation. The intersegmental muscles in the hawkmoth, Manduca sexta, are hormonally committed to degenerate shortly after eclosion (emergence of the moth from the pupa). This programmed neuromuscular cell death is accompanied by a dramatic increase of polyubiquitin gene expression which can be suppressed experimentally by 20-hydroxyecdysone which delays degeneration in this system [98,99]. The increased expression of the polyubiquitin gene occurs without apparent activation of the heat-shock response in the tissues. The observations support the notion that an increased requirement for ubiquitin may be a common element in programmed cell death, presumably reflecting a need for enhanced proteolysis to degrade internal organeUes and proteins during the degenerative process. A complete understanding of the regulatory processes in this

type of developmental situation may have considerable implications for diseases like amyotrophic lateral sclerosis where essentially complete destruction of spinal cord anterior horn neurones and specific brain motor cortex neurones can occur (section II). Protein ubiquitination linked to chaperoning and catabolic events could occur coincidentally during the extensive morphological reorganisation which occurs during cell morphogenesis in early embryonic development. Recently, extensive cytoplasmic protein ubiquitination has been shown to occur in the lens of the eye, myotome and notocord in early chicken embryos [100] and correlates precisely with cell differentiative transitions. The changes in protein ubiquitination appear to be associated with similar increases in the expression of a B crystallin in these tissues. it can be seen that the utilisation of ubiquitin is increased both during programmed cell death and embryonic morphogenesis, in both models extensive intracellular reorganisation takes place. These events are controlled hormonally and by morphogenetic factors. There is no reason to exclude the possibility that ubiquitin and cooperating proteins are involved in morphogenetic changes imposed on cells by some disease process: this is presumably why characteristic changes in patterns of ubiquitination are seen in some chronic degenerative and viral diseasts. Programmed development of plants, particularly in the tissues of the vascular system, involves the degradation of proteins and organelles [101]. Ubiquitin is linked to target proteins by an isopeptide bond between the carboxyl-terminal glycin¢ residue of ubiquitin and the e-amino group of a lysine residue. Multiple ubiquitination of target proteins by additional ubiquitin moieties added sequentially to the first ubiquitin linked to the target protein appears to be required for non-lysosomal ubiquitin-dependent proteolysis (section IVB). Each additional ubiquitin molecule is attached via the e-amino group of lysine-48 of the preceding ubiquitin molecule. A mutant ubiquitin molecule with the lysine at position 48 replaced by arginine cannot participate in multi-ubiquitination and is an inhibitor in vitro of ubiquitin-dependent proteolysis [68]. Recently, the generation of transgenic plants expressing multiple copies of the mutated ubiquitin has resulted in developmental abnormalities including leaf curling, vascular tissue alterations and necrotic lesions in leaves, and transgenie calluses expressing mutated polyubiquitin genes have an abnormal growth response in the presence of canavanine [102]. The experiments are based on the principle that mutant ubiquitin molecules compete with endogenous wild-type ubiquitin molecules thus restricting multiple ubiquitination and proteolysis. These studies show that ubiquitin is necessary for controlled protein catabolism in plant development. The combined recent experimentation indicates that

154 ubiquitin has an important role in animal and plant development. VII. Ubiquitin cross-reactive protein Interferon fl (IFN-fl) has been shown to induce a 15 kDa protein homologous to ubiquitin in cells [ 103-105]. The protein cross-reacts with antiserum to ubiquitin and has thus been named the ubiquitin cross-reactive protein. Tumour necrosis factor can enhance the induction by INF-/] of the ubiquitin cross-reactive protein in some cells. The inductive synergism correlates with the establishment of an anti-viral state in these cells [106]. The function of the ubiquitin cross-reactive protein is not known. Given that the ubiquitin system is pivotal to the cell stress response [107,108] it might be expected that some aspect of the cell response to cytokines could involve ubiquitin or, as it turns out, a ubiquitin crossreactive protein. The precise functions of the ubiquitin cross-reactive protein must be established before we can understand why cytokines increase transcription of the gene for this protein. The ubiquitin-like protein may confer resistance of the cell to viral infection. Presumably the ubiquitin cross-reactive protein is covalently linked to some target proteins in the cell response to cytokines. The conjugation of the ubiquitinlike protein may then be involved in the generation of the anti-viral state in the cell. it is interesting to speculate that if protein ubiquitination evolved, in part, for the elimination of proteins by a catabolic route then gene duplication and divergence may have produced a ubiquitin-like protein with a ubiquitin-related function, which is specifically transcribed in response to cytokines and is involved in resistance to viral infection. Clearly, if ubiquitin evolved to assume roles in normal protein catabolism and also in the elimination of abnormal and damaged proteins [2] then ubiquitin or some divergent protein might be expected to have a role in the elimination of foreign proteins, including viral proteins, from the cell. Ubiquitin is found conjugated to proteins in several types of viral particle (section V): it will be interesting to discover if the ubiquitin cross-reactive protein can be similarly conjugated in viraily infected cells. VIII. Evolutionary perspective on ubiquitin and the eukaryotic cell Ubiquitin is found only in eukaryotic cells where it shows remarkable conservation from lower to higher organisms [117]. The protein certainly has roles in the nucleus through histone ubiquitination and in the cytoplasm in protein catabolism. The protein degradative functions have probably been adapted as part of a cytoprotective system operative against modified, dam-

aged, foreign and viral proteins. It is worth dwelling for a moment on chickens and eggs, i.e., which came first! Such hypotheses are limited by a lack of understanding of the receptor systems which recognise singly or multiply ubiquitinated proteins. Clearly such receptors will have functions vital for our understanding of the roles of protein ubiquitination in cells. The fact that a proportion of histones H2A and H2B are ubiquitinated, that one ubiquitin conjugating enzyme (E~) is a yeast cell cycle protein (CDC 34) and that another E 2 is a yeast radiation repair enzyme (RAD 6) implies that protein ubiquitination has key roles in cell division and nuclear functions which appear ostensibly unrelated to protein degradation [117]. However, the recent demonstrations that the cell cycle is regulated by the ubiquitin-dependent degradation of cyclin [73] and that the in vitro degradation of nuclear oncoproteins (N-myc, cmyc, c-fos, p53 and EIA) is both ATP-dependent and inhibited by antiserum to the ubiquitin activating enzyme E I [74] suggests that a catabolic role for ubiquitin in nuclear function may have been evolved early in the life process. The extreme importance of precise control of cell division and transcription led to the evolution of regulatory protein molecules with very rapid turnover and small pool sizes so that the concentration of these molecules could be rapidly changed in very short periods of time. Mutation of some of these regulatory proteins in the nucleus is associated with cell transformation and tumour progression. This ubiquitin-dependent degradation system would have been exported to the cytoplasm to facilitate control of other protein molecules such as regulatory enzymes in metabolic pathways. But what of long lived proteins? These have been considered to be generally degraded in the lysosomal system, which until recently has been thought to operate in a ubiquitin-independent manner. The newer information which shows that ubiquitin-protein conjugates are enriched in the lysosomal system of several cell types [58,61,63,64] and that protein ubiquitination is necessary for the lysosomai degradation of some proteins in stressed cells [66] begs the question of how this connection between ubiquitin and the lysosomal system may have evolved. An answer may come when we fully understand ubiquitination of histones in relation to chromatin structure and the nuclear envelope. Currently accepted models of the organisation of the nuclear interphase lamina and the relationship between the lamina and the reversible mitotic disassembly of the nuclear envelope [109] show insoluble chromatin fibres binding through lamins (lamin B) to the double membrane nuclear envelope (Fig. 3A-i). The lamins are members of the intermediate filament family of gene products [43]. A key unknown is whether ubiquitinated proteins may be involved in the interaction between chromatin fibres, lamins and the double

155 membrane of the nuclear envelope, e.g., as suggested in Fig. 3A-ii. it is possible that ubiquitinated histones could contribute to tills interaction. Proteins involved in such an association cGuld then be readily adapted to function in the process of membrane wrapping, which is a feature of autophagosome formation as shown in Fig. 3B. Alternatively, where the ubiquitinated protein is mobile in solution, rather than anchored to an insoluble matrix as in chromatin fibres, then some form of microautophagy could occur; such a process might exploit a ubiquitin-sensitive motor activated to drive surface invagination of lysosome-related vesicles and form multivesicular bodies (Fig. 3C). Multivesicular bodies may well be central to the degradation of certain ubiquitinated proteins [58,61,64]. The situation in the metaphase nucleus when the nuclear envelope is fragmented into double membrane vesicles may have provided the starting point for the evolution of a double membrane wrapping process. There is certainly an association between protein ubiquitination, intermediate filaments and the lysosome system as indicated in chronic degenerative disease (section ll) and in the cell response to some viral proteins (section V). Nuclear lamins may be present in the intranuclear ubiquitihated inclusions (Marinesco bodies) seen in intranuclear hyaline inclusion disease [42].

or foreign proteins and organelles. The purpose of this system is to ~solate and eliminate these noxious structures from the cell: as a cytoprotective mechanism this appears to have evolved in the cell akin perhaps to an 'intracellular immune system'. Other heat shock proteins such as hsp 70 may be involved in this process. it is apparent that ubiquitin has a role in embryonic development. Protein ubiquitination is presumably involved in the reorganisation of cytoplasm that accompanies cell differentiation. Ubiquitin is also necessary for the gross intracellular degradative processes which are consequent upon programmed cell death. Cell elimination is of key importance for a number of developmental morphogenetic changes. An understanding of the molecular details of these processes will no doubt provide further insights into the wide ranging roles of ubiquitin in the life process. As it says in the book 'Ubiquitin' [110]; there is no doubt that ubiquitin is a 'lucky' protein. It is lucky in many ways: lucky for scientific progress, lucky for biomedical scientists and lucky for life! If you have not already done so, why don't you get lucky and look for a role for ubiquitin in your experimental system. As Avram Hershko has said "there is plenty to go round"!

IX. Summary

We would like to thank the Wellcome Trust, The Parkinson's Disease Society of Great Britain and The Motor Neurone Disease Association for support of some of the work described in the review. One of us (R.J.M.) would also like to devote the article to his late sister-in-law, Heather, for her gentleness and kindness and for helping to remind us all of a writing in Wiirtzburg attributed to Martin Luther which reads:

Studies in recent years have shown that ubiquitin has increasingly important functions in eukaryotic cells; roles which were previously not suspected in healthy and diseased cells. The interplay between molecular pathological and molecular cell biological findings has indicated that ubiquitin may be pivotal in the cell stress response in chronic degenerative and viral diseases. Furthermore, the studies have led to the notion that ubiquitination may not only serve as a signal for nonlysosomal protein degradation but may be a unifying covalent protein modification for the major intracellular protein catabolic systems; these can act to identify proteins for cytosolic proteinases or direct intact and fragmented proteins into the lysosome system for breakdown to amino acids. This unifying role could explain why ubiquitin is restricted to eukaryotic cells, which possess extensive endomembrane systems in addition to a nuclear envelope. Protein ubiquitination is a feature of most filamentous inclusions and certain other intraceilular conglomerates that are found in some degenerative and viral diseases. The detection of ubiquitin-protein conjugates is now of great diagnostic importance in these diseases. Protein ubiquitination is not only essential for the normal physiological turnover of proteins but appears to have been adapted as part of an intracellular surveillance system that can be activated by altered, damaged

Acknowledgements

"Wer nicht iebt Wein, Weib und Gesang Der bleibt ein Narr sein Leben lang"

Re~erences I Finley.D. and Varshavsky. A. (1985) Trends Biochcm Sci. I0, 343-347. 2 Hershko,A. (1988)J. Biol.Chem. 263. 15237-15240. 3 Cieehanover,A. and Schwartz.A.L.(1989)TrendsBiochem.Sci. 14, 483-488. 4 Jentsch, S., Seufert. W., Sommer, T. and Reins. H.A. (1990) Trends Biochem. Sci. 15, 195-198. 5 Wilkinson.K.D. (t987) Anti-CancerDrug Des. 2. 21!-229. 6 Ciechanover,A., Heller, H., Elias. S., Haas, A.L. and Hershko, A. (1980) Proc. Natl, Acad. Sci. USA 77, 1365-1368. 7 Ciechanover,A., Heller, H., Katz-Eltzion,C. and Hershko, A. (1981) Proc. Natl. Acad. Sci. USA 78, 761-765. 8 Ciechanover,A., Elias. S., Heller. H. and Hershko. A. (1982)J. Biol. Chem. 257, 2537-2542. 9 Raimport.S.. Dubiel.W. and Muller.M. (1985)FEBSLett. 180. 249-252. 10 Siegelman,M.. Bond, M.W..Gallatin.W.M.,John, T.S., Smith. H.T.. Fried, V.A. and Weissman,1.L (1986) Science 231, 823829.

156 il John, T.S., Gallatin, W.M., Siegelman M., Smith, H.T., Fried, V.A. and Weissman, I.L. (1986) Science 231.45-50. 12 Goldknopf, I.L. and Busch, H. (1977) Prec. Natl. Acad. Sci. USA 74, 864-868. 13 Mealier, R.D., Yasuda, H., Hatch, C., Banner, W.M. and Bradbury, E.M. (1985) J. Biol. Chem. 260, 5147-5153. 14 Nickel. B.C., Allis, C.D. and Davie, J.R. (1989) Biochemistry 28, 958-963. 15 Jentsch, S., McGrath, J. and Varshavsky, A. (1987) Nature 329, 131-134. 16 Goebl, M.G., Yochem, J., Jentsch, S., McGrath, J.P., Vat* shavsky, A. and Byers, B. (1988) Science 241, 1331-1335. 17 Claude, A. (1938) Prec. Sac. Exp. Biol. Mad. 39, 398-403. 18 Lowe, J., Blanchard, A., Morrell, K., Lennox, G., Reynolds, L., Billett, M., Landon, M. and Mayer, R.J. (1988) J. Pathol. 155, 9-15. 19 Mori, H., Kondo, J. and lhara, Y. (1987) Science 235, 1641-1644. 20 Perry, G., Friedman, R., Shaw, G. and Chau, V. (1987) Proc. Natl. Acad. Sci. USA 84, 3033-3036. 21 Cole, G.M. and Timiras, P.S. (1987) Neurosci. Lett. 79, 207-212. 22 Wischik, C.M., Novak, M, Thogersen, H.C., Edwards, P.C., Runswick, M.J., Jakes, R., Walker, J.E., Milstein, C., Roth, M. and Klug, A. (1988) Proc. Natl. Acad. Sci. USA 85, 4506-4510. 23 Collier, N.C. and Schlesinger, M.J. (1986) J. Cell Biol. 103, 321-325. 24 Earl, R.T., Mangiapane, E.H., Billett, E.E. and Mayer R.J. (1987) Biochem. J. 241,809-815. 25 Galloway, P.G., Grundke-lqbal, 1., Iqbal, K. and Perry, G. (1988) J. Neuropathol. Exp. Neurol. 47, 654-63. 26 Kuzuhara, S., Mori, H., lzumiyama, N., Yoshimura, M. and lhara, Y. (1988) Acta Neuropathol. 75, 345-353. 27 Love, S., Saitoh, T., Quijada. S., Cole, G.M. and Terry, R.D. (1988) J. Neuropathol. Exp. Neurol. 47, 393-405. 28 Kosaka,K. (1978) J. Neurol. Neurosurg. Psych. 42, 127-134. 29 Lennox, G., Lowe, J., Landon, M., Bvrne. J.. Mayer. R.J. and Godwin-Austen, R.B. (1989) J. Neural. Neurosurg. Psych. 52, 1236-1247. 30 Lowe, J., Morrell, K., Lennox, G., Landon, M. and Mayer, R.J. (1988) Neuropathol. Appl. Neurobiol. 15, 45-53. 31 Leigh, P.N., Anderton, B.H., Dodson, A., Gallo, J.-M., Swash, M. and Power, D.M. (1988) Neurosci. Lett. 93, 197-203. 32 Lowe, J., Lennox, G., Jefferson, D., Morell, K., D., M., Gray, T., Landau, M., Doherty, F.J. and Mayer, R.J. (1988) Neurosci. Lett. 94, 203-210. 33 Lowe, J., Aldridge, F., Lennox, G., Doherty, F., Jefferson, D.. Landau, M. and Mayer, R.J. (1989) Neurosci. Lett. 105, 7-13. 34 Bunina, T.L. (1962) Ah. Neuropat. Psik. hit. Korsakov. 62, 1293-1299. 35 Heggie, P., Burdon, T., Lowe, J., Landau, M., Lennox, G., Jefferson, D. and Mayer, R.J. (1989) Neurosci. Lett. 102, 343348. 36 Wilkinson, K.D., Lee, K., Deshpande, S., Duerksen-Hughes, P., Boss, J.M. and Pohl, J. (1989) Science 246, 670-673. 37 Lowe, J., McDermott, H., Landau, M., Mayer, R.J. and Wilkinson, K.D. (1990) J. Pathol. 1610 153-160. 38 lwaki, T., Kume-lwake, A. and Goldman, J.E. (1990) J. Histochem. Cytochem. 38, 31-39. 39 l.owe, J., McDermott, H., Pike, !., Spendlove, l., Landau, M. and Mayer, R.J. (1991) J. Pathol., in press. 40 Lennox, G., I.owe, J., Morrell, K., Landau, M. and Mayer, R.J. (1988) Neurosci. Lett. 94, 211-217. 41 Mann, D.M.A., Prinja, D., Davies, C.A., lhara, Y., Delacourte, A., Defosscz, A., Mayer, R.J. and Landau, M. (1989) J. Neural. Sci. 92, 247-260. 42 Bancher, C. Lassman, H., Budka, H., Jellinger, K., Grundkelqbal, i., iqbal, K., Wiche, G., Seitelberger, F. and Wisniewski, H.M. (1989) J. Neuropathol. Exp. Neural 48, 81-93.

43 McKeon, F.D., Kirschner, M.W. and Caput, D. (1986) Nature 319, 463-468. 44 Cataldo, A.M., Thayer, C.V., Bird, E.D., Wheelock, T.R. and Nixon, R.A. (1990) Brain Res. 513, 181-192. 45 Lowe, J., McDermott, H., Kenward, N., Landau, M., Mayer, R.J., Bruce, M., McBride, P., Somerville, R.A. and Hope, J. (1990) J. Pathol. 162, 61-66. 46 Boellaard, J., Schlote, W. and Tateishi, T. (1989) Acta Neuropathol. 78, 410-418. 47 Suenaga, T., Hirano, A., Llena, J.F., Ksiezakreding, H., Yen, S.H. and Dickson, D.W. (1990) Ann. Neural. 28, 174-177. 48 Lennox, G., Lowe, J., Morrell, K., Landau, M. and Mayer, R.J. (1989) J. Neural. Neurosurg. Psych. 52, 67-71. 49 Dickson, D.W., Crystal, H. and Mattiace, L.A. (1989) Acta Ncuropathol. 78, 572-584. 50 Hansen, L., Salmon, D., Galasko, D. and Masliah, E. (1990) Neurology 40, 1-8. 51 Perry, R.H., Irving, D., Blessed, G., Fairburn, A. and Perry, E.K. (1990) J. Neural. Sci. 95, 119-139. 52 Mayer, R.I., Lowe, J., Lennox, G., Landon, M., MacLennan, K. and Doherty, F.J. (1989) Biochem. Sac. Syrup. 55, 193-201. 53 Lowe, J. and Mayer, RJ. (1990) Neuropathol. Appl. Neurobiol. 16, 281-291. 54 Magnusson K. and Wieloch, T. (1989) Neurosci. Lett. 96, 264270. 55 Vass, K., Welch, W.J. and Novak, T.S.Jr., (1988) Acta New ropathol. 77, 128-135. 56 Zhaung, Z.P. and McCauley, R. (1989) J. Biol. Chem. 264, ! 4594-14596. 57 Mayer, R.J., Lowe, J., Lennox, G., Doherty, F. and Landon, M. (1989) in Alzheimer's Disease and Related Disorders (lqbal, IC, Wisniewski, H.M. and Winblad, B., eds.), pp. 809-818, Alan R. Liss, New York. 58 Doherty, F.J., Osborn, N.U., Wassell, J.A., Heggie, P.E., Laszlo, L. and Mayer, R.J. (1989) Biochem. J. 263, 47-55. 59 Doherty, F.J., Laszlo, L., l.owe, J., Lennox, G., Landon, M. and Mayer, R.J. (1989) in Current Trends in the Study of Intracellular Protein Degradation. (Knecht, E. and Grisolia, S., eds.) pp. 255-274, Springer International and University of Basque Country, Berlin and Vizcaya. 60 Schwartz, A.L., Ciechanover, ~., Brandt, R.A. and Geuze, H.J. (1988) EMBO J. 7, 2961-2966. 61 Laszlo, L., Doherty, F.J., Osborn, N.U. and Mayer, R.J. (1990) FEBS Lett. 261,365-368. 62 Doherty, F.J., Osborn, N.U., Wassell, J.A., Laszlo. L. and Mayer. R.J. (1989) Biochem. Sac. Trans. 17, 171-172. 63 Laszlo, L., Doherty, F.J, Watson, A., Self, T., Landon, M., I..owe. J. and Mayer, R.J. (1991) FEBS Lett. 279. 175-178. 64 Laszlo, L., Tuckwell, J., Self, T., i.owe, J., Landon, M., Smith, S., Hawthorne, J.N. and Mayer, R.J. (1991) J. Pathol. 164, in press. 65 Hershko. A. (1985) Prog. Clin. Biol. Res. 180, 11-16. 66 Gropper, R., Brandt, R.A., Elias, S., Bearer, C.F., Mayer, A., Schwartz, A.L. and Ciechanover. A. (1991) J. Biol. Chem., in press. 67 Bachmair, A. and Varshavs~'. A. (1989) Cell 56, 1019-1032. 68 Chau, V., Tobias. J.W., Bachmair, A., Marriott, D., Ecker, D.J., Gonda, D.K. and Varshavsky, A. (1989) Science 243, 1576-1583. 69 Gonda, D.K., Bachmair, A., Wunning, 1., Tobias, J.W., Lane, W.S. and Varshavsky, A. (1989)J. Biol. Chem. 264, 16700-16712. 70 Johnson, E.S., Gonda, D.K. and Varshavsky, A. (1990) Nature 346. 287-291. 71 Chen, Z. and Pickart, C.M. (1990) J. Biol. Chem. 265, 2183521842. 72 Jabben. M., Shanklin, J. and Vierstra, R.D. (1989) J. Biol. Chem. 264, 4998-5005.

157 73 Glotzer, M., Murray, A.W. and Kirschner, M.W. (1991) Nature 349, 132-138. 74 Ciechanover, A.. DiGuiseppe. J.A., Bercovich. B., Orian, A., Richter, J.D.. Schwartz. A.L. and Brodeur, G.M. (1991) Proc. Natl. Acad. Sci. USA 88, 139-143. 75 Shaeffer, J.R. (19881 J. Biol. Chem. 263, 13663-13669. 76 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, U., Fried, V. and Williams, L.T. (1986) Nature 323, 226-232. 77 Leung, D.W., Spencer, S.A., Cachianes, G., Hammonds. R.G., Collins, C., Henzel, W.D., Barnard, R., Waters, M.J. and Wood, W.I. (1987) Nature 330, 537-543. 78 St John, T., Gallatin, W.M., Siegelman, M. Smith. H.T., Fried, V.A. and Weissman, I.L. (1986) Science 231,845-849. 79 Ball, E., Karlik, C.C., Beall, CJ., Saville, D.L, Sparrow, J.C., Bullard, B. and Fyrberg, E.A. (1987) Cell 51,221-8. 80 Carlson, N. and Rechsteiner, M. (1987) J. Cell Biol. 104, 537-46. 81 Goldknopf, I.L., French, M.F., Daskal, Y. and Busch, H. (1978) Biochem. Biophys. Res. Commun., 84, 786-793. 82 Thorne, A.W., Sautiere, P., Briand, G. and Crane-Robinson. C. (1987) EMBO J. 6, 1005-1010. 83 Haas, A.L., Bright, P.M. and Jackson, V. (1988) J. Biol. Chem. 263, 13268-13275. 84 Berleth, E.S. and Pickart, C.M. (1990) Biochem. Biophys. Res. Commun. 171,705-710. 85 Pickart, C.M. and Vella, A.T. (1988)J. Biol. Chem. 263, 1507615082. 86 Haas, A., Reback, P.M., Pratt, G. and Rechsteiner. M. (1990)J. Biol. Chem, 265, 21664-21669. 87 Meyers, G., Rumenapf, T. and ThieL H.J. (1989) Nature 341, 491. 88 Guarino, L.A. (1990) Proc. Natl. Acad. Sci. USA 87, 409-413. 89 Kemp, L.M. and Latchman, D.S. (1988) Virology 166, 258-261. 90 Dunigan. D.D., Dietzgen, R.G., Schoelz, J.E. and Zaitlin, M. (1988) Virology 165, 310-312. 91 Hazelwood, D. and Zaitlin, M. (1990) Virology 177, 352-356. 92 Putterman, D., Pepinsky, R.B. and Vogt, V.M. (1990) Virology 176, 633-637. 93 Dice, J.F. (1990) Trends Biochem. Sci. 15, 305-309. 94 Moorthy, R. and Thorley-Lawson, D.A. (1990) J. Virol. 64, 829-837. 95 Mann, K.P., Staunton, D. and Thorley-Lawson, D.A. (1985) J. Virol. 55, 710-720.

96 Longknecker, R. and Kieff, E. (1990) J. ViroL 64, 2319-2326. 97 Glenner, G. (1989) Annu. Rev. Med. 40. 45-51. 98 Schwartz, L.M., Kosz, L. and Kay, B.K. (1990) Proc. Natl. Acad. Sci. USA 87, 6594-6598. 99 Schwartz, L.M., Myer, A., Kosz, L., Engelstein, M. and Meier, C. (1990) Neuron 5, 411-419. 100 Scotting, P., McDermolt, H., [..owe. J. and Mayer. R.J. (1991) FEBS Left., in press. 101 Woolhouse, H.W. (1984) in Cell Ageing and Cell Death. (Davies, l. and Sigee, D.C., eds.), pp. 123-153, Cambridge University Press, Cambridge. 102 Bachmair, A., Becker, F, Masterson, R.V. and Schell, J. (1990) EMBO J. 9, 4543-4549. 103 Korant, B.D., Blomstrom, D.C., Jonak, G.J. and Knight, E. (1984) J. Biol. Chem. 259, 14835-14839. 104 Knight, E., Fahey, D., Cordova, B., Hillman, M., Kutny, R. and Reich, N. (1988) J. Biol. Chem. 263, 4520-4522. 105 Haas, A.L, Ahrens, P., Bright, P.M. and Ankel, H. (1987) J. Biol. Chem. 262, 11315-11323. 106 Ahrens, P.B., Besancon, F., Memet, S. and Ankel, H. (1990) J. Gen. Virol. 71, 1675-1682. 107 Finley, D., Ciechanover, A. and Varshavsky, A. (1984) Cell 37, 43-55. 108 Bond, U. and Schlesinger, MJ. (1985) Mol. Cell. Biol. 5, 949-56. 109 Gerace, L., Comeau, C. and Benson, M. (1984) J. Cell Sci., Suppl. 1, 137-160. 110 Rechsteiner, M. (1988) Ubiquitin, Plenum Press, New York. Ill Chirico, W.J., Waiters M.G. and Blobel, G. (1988) Nature 333, 805-810. 112 Kang, P-J., Ostyermann, J., Shilling, J., Neupert, W. and Craig, E.A. (1990) Nature 348, 137-143. !13 Stahl, N., Borchelt, D.R., Hsiao, K. and Prusiner. S.B. (1987) Cell 51, 229-240. 114 Goate, A., Chartier-Harlin, M-C., Mullan, M., Brown, J., Crawford, F., Fidani, L , Giuffra, L., Haynes, A., Irving, N., James, L., Mant. R., Newton, P., Rooke, K., Roques, P., Talbot, C., Pericak-Vance, M., Roses, A., Williamson, R., Rossor, M., Owen, M. and Hardy, J. (1991) Nature 349, 704-7116. 115 Hsiao, K.K., Scott, M., Foster, D., Groth, D.F., DeArmond, SJ. and Prusiner, S.B. (1990) Science 250, 1587-1590. 116 Migheli, A., Attanasio, A., Vigliani, M.C. and Schiffer, D. (1991) Neurosci Lett. 121, 55-58. 117 Jentsch, S., Seufert, W. and Hauser, H.-P. (1991) Biochim. Biophys. Acta 1089, 127-139.