Illuminating the ubiquitin/proteasome system

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Review

Illuminating the ubiquitin/proteasome system

Florian A. Salomons, Klàra Ács, Nico P. Dantuma⁎

Department of Cell and Molecular Biology, Karolinska Institutet, S-17177 Stockholm, Sweden

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +46 8 313529.E-mail address: nico.dantuma@ki.se (N.P. DAbbreviations: CFP, cyan fluorescent protein;

red fluorescent protein; SOD1, superoxide dism

0014-4827/$ – see front matter © 2010 Elseviedoi:10.1016/j.yexcr.2010.02.003

A B S T R A C T

Article Chronology:

Received 22 December 2009Accepted 1 February 2010Available online 10 February 2010

The ubiquitin/proteasome system (UPS) is responsible for the regulated processive degradation ofproteins residing in the cytosol, nucleus, and endoplasmic reticulum. The two central players areubiquitin, a small protein that is conjugated to substrates, and the proteasome, a large multi-subunit proteolytic complex that executes degradation of ubiquitylated proteins. Ubiquitylationand proteasomal degradation are highly dynamic processes. During the last decade, many

researchers have started taking advantage of fluorescent proteins, which allow studying thedynamic nature of this system in the context of its natural environment: the living cell. In thisreview, we will summarize studies that have implemented this approach to examine the UPS anddiscuss novel insights in the dynamic organization of the UPS.

© 2010 Elsevier Inc. All rights reserved.

Keywords:

Proteolysis

Ubiquitin26S proteasomeGreen fluorescent protein

Contents

General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1289Fluorescent ubiquitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1290Fluorescent proteasome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1292Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1294

General introduction

Proteolysis is crucial for maintaining the quality of the cellularproteome. Among the various proteolytic systems in eukaryoticcells, the ubiquitin/proteasome system (UPS) is responsible for thecontrolled processive degradation of proteins residing in thenucleus, cytosol, and endoplasmic reticulum. This requires thecoordinated actions of a large number of proteins that are involved

antuma).GFP, green fluorescent protutase-1; Ub, ubiquitin; UPS

r Inc. All rights reserved.

successively in the recognition of substrates, the tagging ofsubstrates with poly-ubiquitin chains, and the destruction ofpoly-ubiquitylated substrates [1]. The recognition and tagging aremediated by a large group of ubiquitylation enzymes thatconjugate poly-ubiquitin chains to substrates [2], while the finaldestruction is executed by a large multi-subunit proteolyticcomplex known as the proteasome [3]. Regulated proteolysis bythe UPS plays a major role in a large variety of cellular processes

ein; NER, nucleotide excision repair; PAGFP, photo-activatible GFP; RFP,, ubiquitin/proteasome system; YFP, yellow fluorescent protein

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that include cell cycle progression, signal transduction, DNA repair,antigen presentation, and protein quality control [1]. Alterations inthe UPS are associated with a plethora of human pathologies, likeinflammation, malignancies, muscle wasting disorders, neurode-generative diseases, and viral infections [4]. In many cases, the roleof UPS dysfunction in the pathophysiology remains enigmatic.Interestingly, the UPS has also emerged as an anti-cancer drugtarget underscoring its therapeutic potential [5].

Although we have learned a great deal about the subcellulardistribution of components of theUPS by classic immunohistochem-ical analysis of fixed samples, this methodology has offered littleinsight in the dynamic behavior of the UPS and the functional statusof this proteolytic system in health and disease. During the lastdecade, a number of groups have generated fluorescently taggedUPSproteins to study this complex system in living cells. These studiesrevealed new features of the UPS in diverse cellular processes andshed light on how the UPS is affected during environmental andphysiological conditions that threaten the cellular proteome. In thisreview, we will summarize studies that were performed withfluorescent-labeled proteins that allow monitoring the two mainplayers in the UPS: ubiquitin and the proteasome.

Fluorescent ubiquitin

Covalent linkage of the C terminus of ubiquitin to a lysine residue insubstrates, a process knownas ubiquitylation, has a central regulatoryrole in a variety of cellular mechanisms. Besides its canonical role intargeting proteins for proteasomal degradation, ubiquitylation canalso change the fate of a protein in very different ways. Non-proteolytic functions of ubiquitin are important in, for example,chromatin remodeling, DNA repair, endocytosis, signal transduction,and transcription [6]. To warrant such a wide functional range,ubiquitinmodifications come inmanydifferent varieties: conjugationof one single ubiquitin moiety (mono-ubiquitylation), several singleubiquitin moieties (multi-ubiquitylation), and different conforma-tions of ubiquitin chains (poly-ubiquitylation) [7].

Ubiquitylation is executed by a cascade of enzymes, involvingan E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugatingenzyme, an E3 ubiquitin ligase, and in some cases, an E4 chainelongation enzyme [2]. The specificity of this process is ensured byseveral hundred distinct E3 ubiquitin ligases that recognizespecific substrates and, in concert with one of the approximatelytwenty E2 ubiquitin-conjugating enzymes, covalently link ubiqui-tin to the substrate. Ubiquitin itself can be a target for ubiquityla-tion via one of its seven lysine residues creating specific poly-ubiquitin chains. For example, poly-ubiquitin chains linkedthrough the lysine at position 48 in ubiquitin typically targetproteins for proteasomal degradation, while poly-ubiquitin chainswith lysine-63 linkages are, among other things, associated withthe DNA damage response [7]. Mono- or multi-ubiquitylation ismainly involved in endocytosis, multivesicular body formation,and chromatin remodeling [8]. These different ubiquitin conju-gates function as cellular signals and are fundamental for theregulation of protein–protein interactions. Cells contain a largenumber of proteins with ubiquitin-binding domains that recognizeand distinguish different types of ubiquitin modifications andchange the fate or behavior of the ubiquitylated protein [9].

To gain more insight in the dynamic behavior of ubiquitin,several groups employed a chimeric protein consisting of ubiquitin

with an N-terminal fluorescent protein [10–18]. Biochemicalanalysis [19] and fluorescence microscopy [14] confirmed thatthe incorporation and cellular distribution of fluorescent-taggedubiquitin are very similar to endogenous ubiquitin, suggesting thatthe tag does not considerably interfere with the conjugationprocess. These chimeric proteins have been used to study thebehavior of ubiquitin in protein degradation, DNA repair, mem-brane trafficking, and cytokinesis in living cells.

Live cell imaging with green fluorescent protein (GFP)-taggedubiquitin (GFP-Ub) showed that the mobility of ubiquitin in thecytosol and the nucleus is relatively slow compared to theunconjugatable GFP-UbK0,G76V mutant, suggesting that the vastmajority is conjugated to substrates [14] (Fig. 1A). Ubiquitinmobility in the cytosol is predominantly determined by thefraction of large ubiquitin conjugates, while the nuclear immobilepool corresponds with ubiquitylated histones, a major target forubiquitylation in the nucleus. Photo-activatible GFP (PAGFP) is aGFP variant that requires photoconversion by a 405-nm laser lightfor efficient fluorescence. Photo-activation of PAGFP-Ub in a smallnuclear region demonstrated a slow but constant redistributionfrom the activated region to other regions within the cell, whichprobably reflects the constitutive ubiquitylation and deubiquityla-tion of histones [14] (Fig. 1B). This underscores the flexible natureof ubiquitin modifications and illustrates that ubiquitylation anddeubiquitylation enzymes ensure a continuous flux of ubiquitinfrom conjugated forms to the pool of free ubiquitin molecules. Thefinding that a large fraction of the cellular ubiquitin pool is eitherconjugated or in the process of being conjugated, while the levelsof free, uncommitted ubiquitin are relatively small, is consistentwith biochemical observations [19].

Interesting observations were made when GFP-Ub-expressingcells were exposed to various proteotoxic insults such as protea-some inhibition and hyperthermal stress. A major redistribution ofGFP-Ub from the nuclear to the cytoplasmic compartment tookplace, which was accompanied by a reduction in GFP-Ub mobility[14,17]. These changes coincided with the accumulation ofubiquitylated proteasome substrates and a global deubiquitylationof histones as well as a reduction in free ubiquitin [14]. Proteotoxicstress-inflicted redistribution of ubiquitin had a clear effect onchromatin condensation [14] and proteolysis by the 26S protea-some [17]. Based on these findings, it has been postulated thatdifferent ubiquitin-dependent processes are functionally linkedthrough a direct competition for the limited pool of free ubiquitin,which may contribute to the coordination of the cellular responseto proteotoxic stress [20].

In addition to environmental insults, proteotoxic stress can beprovoked by physiological factors such as protein oxidation,aberrant transcription and translation, mutant gene products,and protein unfolding. This can lead to an accumulation andaggregation of aberrant proteins, which can have severeconsequences for the viability of cells. A large variety ofhuman disorders, collectively called conformational diseases[21], are tightly associated with proteotoxic stress and theformation of intracellular inclusions [22]. These inclusions, whichcontain aggregated proteins, often contain ubiquitin, suggestinga possible role for ubiquitin in these pathologies [23]. One of themutant proteins that accumulate in ubiquitin-positive inclusionsis mutant ataxin-1, which is responsible for the inheritableneurodegenerative disorder spinocerebellar ataxia 1. The dy-namic properties of yellow fluorescent protein (YFP)-tagged

Fig. 1 – Fluorescent ubiquitin. (A) Micrographs of living human melanoma cells expressing GFP-Ub. GFP-Ub has a strong granularlocalization in the nucleus, and a diffuse cytoplasmic distribution with a large number of mobile structures. (B) Micrographs ofliving cells as in panel A but expressing themutant ubiquitin GFP-UbK0,G76V, which cannot be conjugated and is diffusely distributedthroughout the cell. (C) False color image of PAGFP-Ub-expressing cell in which PAGFP-Ub has been photo-activated in a smallregion in the cytoplasm. The redistribution of fluorescent PAGFP-Ub is followed in time. Scale bars represent 10 μm.

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ubiquitin (YFP-Ub) have been analyzed in ataxin-1 inclusions[10]. It was shown that the YFP-Ub in the inclusions hasextremely slow exchange rates, indicating that ubiquitin isirreversible conjugated to aggregated mutant ataxin-1 or otherproteins residing in inclusions.

Fluorescent-tagged ubiquitin also accumulated in the largecytoplasmic inclusions called DALIS (dendritic cell aggresome-likeinduced structures), which are specifically found in dendritic cellsand believed to be important for antigen presentation [12]. Similarcellular structures were also formed upon treatment of dendriticcells with puromycin, which causes an accumulation of improperlytranslated proteins. Unlike the inclusion bodies that are formed inresponse to aggregation-prone proteins (such asmutant ataxin-1),puromycin-induced inclusions in dendritic cells contained ubiqui-tylation enzymes and constituently recruited ubiquitin, suggestingthat direct ubiquitylation is occurring within the DALIS andunderscoring that these structures are different from the inclusionobserved in conformational diseases [12].

Regulation of DNA repair and the DNA-damage response byubiquitylation has been reported in a number of publications [24].A serendipitous finding in a study with GFP-Ub led to theidentification of the core histone H2A as a new target forubiquitylation in nucleotide excision repair (NER) [15]. It wasfound that local exposure of the nucleus to 488-nm laser lighttriggered a local accumulation of GFP-Ub. Detailed analysis of thisphenomenon showed that histone H2A is rapidly ubiquitylated inresponse to photolesions in the DNA in an NER-dependent fashion.More recently, a similar ubiquitylation response to double-strand

breaks has been reported [25], and a subsequent study revealedthat the H2A ubiquitylation in response to photodamage anddouble-strand breaks shares many features and involves similarubiquitylation enzymes [18].

It has been demonstrated with GFP-Ub that a dramaticrelocalization of the intracellular ubiquitin occurs during cytoki-nesis in mammalian cells when the bulk of the ubiquitin firstassociates with the midbody microtubules followed by sequestra-tion of ubiquitin on the midbody ring itself [16]. Photobleachingexperiments showed that there was little exchange between themicrotubule-localized ubiquitin and the remaining intracellularubiquitin while the ubiquitin at the midbody ring was rapidlyexchanged, suggesting that this pool is subject to constitutiveubiquitylation/deubiquitylation cycles. This process is mediatedby the E3 ubiquitin ligase BRUCE, which is an anti-apoptoticprotein that localizes on vesicular structures [26].

Compared to degradation of protein substrates by the 26Sproteasome and histone ubiquitylation, the fraction of ubiquitininvolved in processes like endocytosis and vesicular trafficking isrelatively small and therefore more challenging to monitor bymeans of fluorescent-tagged ubiquitin. However, this can bepartially circumvented by creating chimeric membrane proteinsthat are directly fused to GFP-Ub. In this way internalization andtrafficking of plasma membrane proteins towards endosomal andlysosomal structures can be followed in living cells. Studies usingthis approach showed that fluorescent ubiquitin can be used tofollow the fate of mono-ubiquitylated membrane proteins andallowed functional analysis of ubiquitin-sorting receptors [11,13].

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Fluorescent proteasome

The 26S proteasome is a large proteolytic complex consisting ofa 20S core particle and one or two 19S regulatory particles (Fig.2A). The 20S core particle consists of four rings, each containingseven subunits. For simplicity, we will use throughout thisreview the unified nomenclature for the proteasome subunits[3,27]. Each of the two outer rings is composed of seven uniqueα subunits, while the two inner rings each consist of sevenunique β subunits [28] (Fig. 2B). The proteolytic activities residein the 20S core particle where the β1, β2, and β5 subunits areresponsible for the caspase-like, trypsin-like, and chymotrypsin-like activities, respectively. In the mammalian proteasome, thesecatalytic active subunits can be substituted by the β1i, β2i, andβ5i subunits, which are induced by interferon γ and importantfor MHC class-I antigen presentation. The 19S regulatory particlecan be divided in the base complex that consist of six AAA-ATPases (Rpt1–6) and four non-ATPase subunits (Rpn1, 2, 10,13), and the lid complex consisting of at least nine non-ATPasesubunits [29] (Fig. 2B).

Labeling proteasome subunits with fluorescent proteins offersthe possibility to visualize proteasome distribution in living cellsand analyze its dynamics using fluorescent bleaching or photo-activation techniques but some considerations should be takeninto account. First of all, the fluorescent label should not interfere

Fig. 2 – Fluorescent proteasome. (A) Schematic presentation of theα- and two β-rings. The 19S regulatory particle consists of a lid anfluorescent-tagged subunits of the 19S lid and base complexes, andin the 19S lid are abbreviated in the image as ‘N’ and ‘T’, respectivecorresponding references are listed. (C) Micrograph of a living humsubunit, α3-GFP, which has a diffuse distribution throughout the c

with the activity and/or incorporation of the subunits into the 26Sproteasome. Unincorporated or non-functional subunits can leadto alterations in protein degradation and may be toxic for cells.Furthermore, unincorporated tagged subunits will lead to incor-rect conclusions regarding localization and dynamics of the 26Sproteasome. Another critical point is that the fluorescently taggedsubunits should not interfere with the interaction between the 26Sproteasome and proteasome-binding proteins like, for example,deubiquitylating enzymes, substrate shuttling factors, and poly-ubiquitin chains.

Several studies have used fluorescent-tagged proteasomesubunits to gain insight in the dynamic behavior of the complex(Fig. 2B). The distribution and dynamics of fluorescent 26Sproteasome were first studied with a GFP-tagged β1i subunit inimmunoproteasomes by Reits and co-workers [30]. They foundthat the mammalian proteasome is homogenously distributedthroughout the cytoplasm and nucleoplasm. Using fluorescencebleaching techniques, they found that the proteasome enters thenucleus via two pathways: (i) slow active unidirectionaltransport of pre-formed proteasomes from the cytoplasm tothe nucleus and (ii) free diffusion, which occurs only duringmitosis when the nuclear envelope is disintegrated [30]. Withthe GFP-tagged α3 subunit (Fig. 2C), it was shown thatproteotoxic stress does not change the mobility of proteasomeswhereas it causes a dramatic change in the diffusion rate ofpoly-ubiquitylated proteins [14].

26S proteasome. The 20S proteolytic core consists of twod a base complex. (B) Schematic presentation of publishedthe 20S core α- and β-ring complexes. The Rpn and Rpt subunitsly. The fluorescent-labeled subunits are highlighted, andan melanoma cell expressing a fluorescent-tagged 20S coreytoplasm and nucleoplasm. Scale bar represents 10 μm.

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The GFP-tagged 20S core subunit α4 and the 19S basesubunit Rpt1 were used to analyze the cellular distribution of the26S proteasome during the interphase and mitosis of thebudding yeast Saccharomyces cerevisiae [31]. Both Rpt1-GFPand α4-GFP localized predominantly at the inner surface of thenuclear envelope and the endoplasmic reticulum. This cellulardistribution was maintained throughout mitosis. Another studyanalyzed the distribution of the 26S proteasome through mitosisand meiosis by monitoring the 19S lid subunit Rpn11-GFP in thefission yeast Schizosaccharomyces pombe [32]. Rpn11-GFP pre-dominantly localized at the nuclear periphery throughoutinterphase and mitosis, similar to Rpt1-GFP and α4-GFP [31].However, the distribution of Rpn11-GFP within the nucleuschanged dramatically during meiosis I and II. During meiosis I,the proteasome became more dispersed while it localized inbetween the separating DNA in meiosis II [32]. Although thesignificance of this distribution is not clear, it could reflectchanges in local proteolytic demands. Redistribution of 26Sproteasome has also been observed upon entry of budding yeastinto the quiescence phase of the cell cycle [33]. Fluorescentlylabeled 20S proteasome subunits α1-GFP, α4-GFP, β2-redfluorescent protein (RFP) and the 19S subunits Rpt6-GFP,Rpn11-GFP and Rpn15-GFP (also known as Sem1) were foundto relocalize from the nuclear compartment into motilecytoplasmic structures. These structures contained fully assem-bled 20S and 19S proteasome particles that rapidly translocatedto the nucleus upon exit from quiescence. The structures werecoined proteasome stress granules since these observationssuggested that they act as transient reservoirs for 26S protea-somes during quiescence [33].

In fission yeast expressing a GFP-Rpn5 subunit, it was foundthat this lid subunit plays an important role in proteasomeassembly and localization [34]. In the absence of Rpn5,proteasomes are incorrectly assembled and localize in thenucleoplasm instead of the inner nuclear envelope. In theabsence of Yin1 (fission yeast ortholog of the mammalian eIF3subunit Int6), Rpn5-GFP was mislocalized and resided through-out the cytosol, indicating that Int6 is involved in the nuclearimport of proteasomes [35]. A large fraction of the proteasomesalso lacked the catalytic β subunits, suggesting that proteasomeassembly is impaired as well. Interestingly, Yin1 interacts withRpn5, and it has been proposed that this binding may beimportant for its regulatory role in proteasome distribution.Further studies using the fluorescent-tagged 19S base and lidsubunits Rpn1-GFP and Rpn5-GFP, respectively, and the 20S coresubunit GFP-α4 showed that the proteasome core, base, and lidcomplexes can be formed and imported independently into thenucleus in budding yeast [36].

Recruitment of 26S proteasomes to specific subcellular struc-tures is eminent in studies on conformational diseases. Immuno-histochemical studies showed that the 26S proteasome localizes inthe ubiquitin-positive inclusions that are a hallmark of many ofthese diseases [23]. Amyotrophic lateral sclerosis is associatedwith aggregation-prone mutants of the Cu/Zn superoxide dis-mutase-1 (SOD1). In neuronal cells expressing cyan fluorescentprotein (CFP)-tagged mutant SOD1, the majority of the β1i-yellowfluorescent protein (YFP) co-localized and tightly associated withthe inclusions formed by the mutant SOD1 [37]. Furthermore, aYFP-based reporter substrate of the UPS accumulated in cells withinclusions, indicative for a general impairment in UPS activity [37].

Fluorescently labeled β1i core subunit was also used to study theproteasome in cells expressing aggregation-prone prolyl-hydrox-ylase isoform PHD3 [38] and polyglutamine-expanded ataxin-1[10]. In nuclear inclusions formed by ataxin-1, photobleachingexperiments revealed that the proteasomes were not irreversiblytrapped in the aggregates [10]. Collectively these studies demon-strated that 26S proteasomes are recruited to large ubiquitylatedaggregates and that, depending on the nature of those structures,this may lead to proteasome dysfunction. Indeed, other studiesconfirm that the properties of these inclusions may be verydifferent depending on the aggregation-prone protein that isresponsible for their formation [37]. Notably, also for buddingyeast, it has been demonstrated with Rpt1-GFP and α4-GFP thatproteasomes are concentrated in a specific protein qualitycompartment that contains misfolded proteins [39].

Interestingly, synaptic activity in neurons triggered redistribu-tion of proteasomes [40]. Upon N-methyl-D-aspartate receptor-dependent depolarization, the 19S base subunit Rpt1-GFP and the20S core subunit α4-mVenus subunits translocate into thedendritic spines coinciding with increased proteasome activity atthe synapses. These data suggest that the spatial enrichment ofproteasomes may be important for local remodeling of the proteincomposition in the synapse.

Recruitment of the 26S proteasome to other subcellularstructures may also reflect a regulatory mechanism. PA28γ is anuclear 11S regulatory particle that can replace the 19S regulatorand stimulate the activity of the 20S core protease during cell cycletransition and proliferation. PA28γ localized with CFP-α7 innuclear speckles, and it was suggested that the proteolyticfunction of this proteasome complex is involved regulatingsplicing factors trafficking within the nucleus [41].

Concluding remarks

Fluorescent labeling of UPS components has given a deeperunderstanding of the behavior of this proteolytic system in livingcells. The recent development of new fluorescent techniques andthe generation of new fluorescent proteins, such as proteins thatundergo photoconversion after exposure to light with a specificwavelength, like PAGFP [42] and mOrange [43], will furtherincrease the power of live cell imaging in studies on the UPS. Theprogress in automated microscopy and data analysis can beemployed in high content screening for factors and/or compoundsthat specifically affect the localization or dynamics of specific UPScomponents. It should be noted that other fluorescent approacheshave been developed to monitor the functional status of the UPS inliving cells such as various fluorescent protein-based UPSsubstrates [44,45] and fluorescent activity probes for labeling ofproteasomes [46,47]. These fluorescent tools combined with newtechniques will hopefully provide us with a more generalunderstanding on the UPS functionality and decipher the role ofthe UPS in human disorders.

Acknowledgments

The research in the Dantuma laboratory is supported by theSwedish Research Council, the Swedish Cancer Society, theNordic Center of Excellence Neurodegeneration, and the

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European Community Network of Excellence RUBICON (Projectno LSHC-CT-2005-018683).

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