Proc. Natl. Acad. Sci. USAVol. 93, pp. 856-860, January 1996Biochemistry

Arabidopsis MBP1 gene encodes a conserved ubiquitinrecognition component of the 26S proteasome

(protein degradation/proteolysis inhibitor/protein interaction/human)

STEVEN VAN NOCKER*t, QUINN DEVERAUXtt, MARTIN RECHSTEINERt, AND RICHARD D. VIERSTRA*§*Department of Horticulture, University of Wisconsin, Madison, WI 53706; and tDepartment of Biochemistry, University of Utah School of Medicine,Salt Lake City, UT 84132

Communicated by Sidney Velick University of Utah, Salt Lake City, UT, November 8, 1995 (received for review July 21, 1995)

ABSTRACT Multiubiquitin chain attachment is a keystep leading to the selective degradation of abnormal polypep-tides and many important regulatory proteins by the eukary-otic 26S proteasome. However, the mechanism by which the26S complex recognizes this posttranslational modification isunknown. Using synthetic multiubiquitin chains to probe anexpression library for interacting proteins, we have isolated anArabidopsis cDNA, designated MBPI, that encodes a 41-kDaacidic protein exhibiting high affinity for chains, especiallythose containing four or more ubiquitins. Based on similarphysical and immunological properties, multiubiquitin bind-ing affinities, and peptide sequence, MBP1 is homologous tosubunit 5a of the human 26S proteasome. Structurally relatedproteins also exist in yeast, Caenorhabditis, and other plantspecies. Given their binding properties, association with the26S proteasome, and widespread distribution, MBP1, S5a,and related proteins likely function as essential ubiquitinrecognition components of the 26S proteasome.

The ubiquitin-dependent proteolytic pathway is a major routefor the elimination of short-lived and dysfunctional proteins ineukaryotes (1, 2). Natural substrates of this pathway includeimportant cellular regulators such as the plant photoreceptorphytochrome A (3), cyclins (4), p53 and c-Jun oncoproteins (5,6), the yeast MATa2 transcriptional regulator and Ga proteinGpal (7, 8), and components of the NF-KB transcriptionalcomplex (9). Degradation of various targets by the ubiquitinpathway is initiated by the covalent attachment of a chain ofmultiple ubiquitins to the target (10). It is accomplished by anATP-dependent cascade of reactions involving the sequentialaction of multiple enzyme families: Els, E2s, and sometimesE3s-the latter two enzymes appear responsible for the spec-ificity of the system. Conjugation results in the formation of aubiquitin-protein adduct where a multiubiquitin chain, linkedinternally via an isopeptide bond through the C-terminalGly-76 of one ubiquitin and a lysine of an adjacent ubiquitin,is attached via its free C-terminal Gly-76 to lysyl s-aminogroups within the target.Once formed, multiubiquitinated proteins are selectively

recognized and then degraded by the 26S proteasome, an'1500-kDa ATP-dependent proteolytic complex (1, 11, 12).The 26S proteasome is composed of two subcomplexes, the20S proteasome (or multicatalytic protease), which containsthe catalytic core of the protease, and the 19S regulatorycomplex, which is responsible for the ATP dependence andspecificity toward ubiquitinated substrates. The 26S protea-some degrades the target protein into small peptides butreleases ubiquitin in a free, functional form. In this way,ubiquitin serves as a reusable signal for protein breakdown.Although many of the -30 potential subunits of the 26S

proteasome have been identified, it is not known which of these

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is the essential factor(s) involved in the specific recognition ofubiquitin-protein conjugates (11, 12). Previous studies showedthat a single 50-kDa polypeptide [designated S5a (subunit 5aof 26S protease)] present in highly purified preparations ofhuman 26S proteasome could bind ubiquitin-lysozyme conju-gates, even after the proteasome preparations were subjectedto SDS/PAGE and immobilized on membranes (13). In lightof the high degree of conservation of 26S subunits betweenplants and vertebrates (14), we exploited this finding to searchfor cDNAs encoding a plant homolog of human S5a by proteininteraction screening of anArabidopsis thaliana cDNA expres-sion library. Here, we describe a gene, designated MBPI,11 thatencodes a protein exhibiting high affinity for multiubiquitinchains in vitro. By various criteria, MBP1 and S5a appear to beinterspecific homologs. These proteins likely act as key mul-tiubiquitin chain recognition components of the 26S protea-some.

MATERIALS AND METHODS

Cloning and Molecular Characterization of ArabidopsisMBPI. Multiubiquitin chains were synthesized from bovineubiquitin (Sigma) as described (15, 16) and individually puri-fied (17). Chains containing between 4 and 7 ubiquitin unitswere pooled and radiolabeled with carrier-free Na125I (0.5mCi; 1 Ci = 37 GBq; Amersham) using lodo-Beads (Pierce).A. thaliana MBP1 was identified in an amplified, A ZAP IIcDNA library prepared with 1-2 kb of poly(A)+ RNA isolatedfrom 3-day-old, etiolated, hypocotyl/cotyledon tissue (ecotypeColumbia) pretreated with ethylene (generous gift of JoeEcker, University of Pennsylvania, Philadelphia). Approxi-mately 2 x 105 recombinant phage were induced and proteinwas transferred to nitrocellulose membranes (18). Membraneswere washed in Tris-buffered saline [TBS; 20mM Tris HCl, pH7.5 (25°C)/0.5 M NaCl], blocked for 2 h in TBS containing 10mg of bovine serum albumin (BSA) per ml, washed briefly inTBS, and incubated for 1 h in TBS containing 10 mg of BSAper ml and 3.5 x 105 cpm per ml of labeled multiubiquitinchains. Phage corresponding to positive plaques were rescuedto phagemids in Escherichia coli strain XL-1 Blue MRF'(Stratagene). Complete nucleic acid sequence was determinedfrom both DNA strands by the dideoxynucleotide chain-termination procedure with single-stranded DNA templates(19). Nucleotide and amino acid sequence manipulationsemployed programs of the University of Wisconsin GeneticsComputer Group software package (20).DNA and RNA Gel Blot Analyses. Genomic DNA and total

RNA were isolated fromArabidopsis, ecotype Columbia, usingQiagen (Chatsworth, CA) nucleic acid purification columns

Abbreviation: S5a, subunit Sa of 26S protease.tS.v.N. and Q.D. contributed equally to this work.§To whom reprint requests should be addressed.IThe sequence reported in this paper has beenGenBank data base (accession no. U33269).

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under conditions suggested by the manufacturer. DNA (2 ,ug)was digested with appropriate restriction endonucleases andfractionated by electrophoresis on 1% agarose gels. RNA (5,tg) was fractionated on 1% agarose/formaldehyde gels. Nu-cleic acids were transferred to Zeta-Probe (Bio-Rad) mem-branes according to Sambrook et al. (18). Probes were labeledwith [32P]dCTP via random priming (18), and hybridizationwas for 8-16 h in 0.25 M Na2HPO4 (pH 7.2; 22°C) containing1 mM Na4EDTA and 7% SDS.Production and Analysis of Recombinant Arabidopsis

MBP1. Arabidopsis MBP1 was synthesized in E. coli using thepET25a expression vector (Novagen) as follows. An Nde I sitewas introduced into the MBP1 cDNA at the translation startusing PCR with a 5' mutagenic oligonucleotide primer (5'-CTGCCTATCGACCATATGGTTCTCGAGGCG-3') and a3' oligonucleotide primer specific to the cloning vector 3' tothe cDNA insert. The PCR product was ligated into the EcoRVsite of pGEM-T (Promega). The resulting plasmid was di-gested with Nde I and a fragment containing the entire MBP1coding sequence and 3' nontranslated region was inserted intothe Nde I site of pET25a. Almost all the PCR product wasreplaced with authentic sequence by exchange with a 1355-bpXho I fragment from the MBP1 cDNA. Recombinant MBP1protein was expressed inE. coli strain BL21(DE3) and purifiedfrom the soluble fraction by a combination of ammoniumsulfate partitioning and gel-filtration and anion-exchangechromatography. Briefly, cells were lysed in 50 mM Hepes, pH8.0 (22°C)/50 mM NaCl/14 mM 2-mercaptoethanol/2 mMNa4EDTA. Protein precipitating between 0% and 30% am-monium sulfate was resuspended in 50 mM Hepes, pH 8.0(22°C)/50 mM NaCl/0.5 mM dithiothreitol and applied to a2.5 x 100-cm BioGel A 1.5M column equilibrated in the samebuffer. Under these conditions, the recombinant protein be-haved as a soluble aggregate with a molecular mass >2 MDa.Protein collected in the excluded fraction was applied to aDE-52 column (Whatman; bed volume, 10 ml) equilibrated inthe same buffer. Bound protein was eluted with 100 mMNa2HPO4/KH2PO4 (pH 8.5; 22°C) containing 0.5 M NaCl. Allprotein purification procedures were carried out at 4°C.MBP1 antibodies were raised in rabbits against highly

purified, hexahistidine-tagged MBP1. A 1355-bp Xho I frag-ment from the MBP1 cDNA in pBluescript II SK (Stratagene)was ligated into the Xho I site of pETlSb. This constructionresulted in an open reading frame in which the two N-terminalamino acids of MBP1 (MV) were exchanged for MGSSHH-HHHHSSGLVPRGSHM. Hexahistidine-tagged MBP1 wasexpressed in E. coli and purified by metal-chelate affinitychromatography using conditions suggested by Novagen.

Purification and Analysis of Human SSa. Human 26Sregulatory complex was purified from human red blood cellsby anion-exchange and gel-filtration chromatography (21).The complex was resolved by one- or two-dimensional gelelectrophoresis and protein was electrophoretically trans-ferred to nitrocellulose membranes. The membranes werestained with Ponceau S to determine relative migration of thesubunits before immunoblot analysis or multiubiquitin chainbinding assays. Immunoblot analysis was performed by theenhanced chemiluminescence (ECL) (Amersham) method.For amino acid sequence analysis of S5a, regulatory complexwas subjected to SDS/PAGE and transferred to nitrocellulose,and the region of the membrane containing S5a was excisedand treated with endoproteinase Lys-C. Recovered peptideswere separated by reversed-phase HPLC and subjected toN-terminal sequence analysis. Binding of 125I-labeled multi-ubiquitin chains to immobilized MBP1 or SSa was assayed asdescribed (13).

RESULTSAs a first step in identifying cellular factors involved inmultiubiquitin chain recognition, we screened anArabidopsis

cDNA expression library for proteins that could physicallyinteract with labeled, free multiubiquitin chains containingbetween 4 and 7 ubiquitin units. Multiubiquitin chains weresynthesized in vitro using the ubiquitin conjugating enzymeTaUBC7, which can catalyze the condensation of ubiquitinmonomers into free chains linked exclusively via Gly-76-Lys-48 (16, 17). We detected and isolated a 1340-bp cDNAdesignated MBP1 (multiubiquitin binding protein). As iden-tified, the MBP1 protein was translationally fused to theN-terminal domain of ,3-galactosidase encoded within AZAP II; this fusion exhibited high affinity for multiubiquitinchains even in the presence of the >10,000-fold molar excessof carrier protein used in the screen.The MBP1 cDNA contained a single long open reading

frame that encoded a protein of 386 amino acids with apredicted pI of 4.3 and a calculated molecular mass of 40,757Da (Fig. 1A). This protein has a short, largely hydrophobicsequence, DXXLALALXXSM, that is repeated twice start-ing at residues 225 and 309; this sequence is followed closelyin each case by a short stretch of charged residues. MBP1 alsocontains an -21-residue, hydrophilic, C-terminal domainconsisting predominantly of charged residues (Fig. 1A). TheMBP1 coding region was bordered by 20 bases of 5' and 159bases of 3' nontranslated sequence (data not shown). Twosequences containing multiple, potential polyadenylylationsignals (25) were found proximal to the 3' end of the cDNA:AATAAAAATT at position -150 (relative to the 3' end ofthe cDNA) and AATTTAACTAA at position -104.Genomic DNA gel blotting under conditions of low strin-gency indicated that MBP1 is a single copy gene in Arabi-dopsis (data not shown). RNA gel blot analysis using theentire cDNA as a probe detected an "1400-base mRNA inallArabidopsis tissues examined, including leaf, stem, flower,and silique; levels of this mRNA were not affected by heatstress (data not shown).To facilitate biochemical characterization of MBP1, recom-

binant MBP1 protein was produced in E. coli using the pETexpression system (26) and purified by conventional chroma-tography (Fig. 2A). Whereas the experimentally determinedpl was in good agreement with the predicted pI (see below),MBP1 migrated anomalously during SDS/PAGE, with anapparent molecular mass of "50 kDa (Fig. 2A). The recom-binant protein was synthesized beginning at the first ATG ofthe open reading frame. This is likely the authentic translationstart site because recombinant MBP1 comigrated during SDS/PAGE with a similarly sized protein present in Arabidopsisextracts that is immunoreactive with MBP1 antisera (Fig. 2C).This 50-kDa immunoreactive species was present in all Ara-bidopsis tissues assayed, including roots, stems, leaves, andflowers (data not shown).Recombinant MBP1 retained the ability to specifically bind

125I-labeled multiubiquitin chains even when subjected toSDS/PAGE and electroelution onto nitrocellulose mem-branes (Fig. 2B). To define the specificity of the MBP1-chaininteraction, purified MBP1 was affixed to nitrocellulose andprobed with a mixture of 125I-labeled multiubiquitin chains;those chains that bound were eluted and their size distributionwas analyzed by SDS/PAGE. Although the original probe waspredominantly ubiquitin monomers and chains of 4 or fewerubiquitins, species that bound to MBP1 were mainly chains of4 or more ubiquitins (Fig. 3C). The affinity of MBP1 for longerchains was especially evident for those species containing -6ubiquitins, which were poorly represented in the original probebut greatly enriched for in the bound fraction (Fig. 3C).A search of the GenBank and Swiss-Prot sequence libraries

revealed that MBP1 is not structurally related to any proteinof known function. However, significant homology was ob-served with potential products of open reading frames fromhuman (54% identity/75% similarity), yeast (45% identity/68% similarity), Caenorhabditis elegans (60% identity/69%

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AMP1 1YeastHumanCaenorbabditisRice

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FIG. 1. (A) Amino acid sequence alignment of Arabidopsis MBP1 with the deduced products of open reading frames from yeast, human,Caenorhabditis, rice, and castor bean. MBPJ was isolated by screening an Arabidopsis cDNA expression library with 125I-labeled multiubiquitinchains. Genes potentially encoding proteins related to MBP1 were identified by a BLAST search of the GenBank sequence library. They includedan open reading frame from the yeast Saccharomyces cerevisiae chromosome VIII [H9998.2; GenBank accession no. U00030 (22)] and partiallysequenced, expressed sequence tags from rice (GenBank accession no. D47537; T. Sasaki, A. Miyao, and K. Yamamoto, unpublished), castor bean[(GenBank accession no. T23240 (23)], human (GenBank accession no. D31409 (24)], and C. elegans (CEESK12; GenBank accession nos. T00848and T00849; W. R. McCombie, J. M. Kelley, L. Aubin, M. Goscoechea, M. G. FitzGerald, A. Wu, M. D. Adams, M. Dubnick, A. R. Kerlavage,J. C. Venter, and C. A. Fields, unpublished). The repeated hydrophobic motif in theArabidopsis MBP1 is underlined. Arrowheads indicate the limitsof reliable sequence for the expressed sequence tags. Numbers refer to amino acid sequence position of MBP1. (B) Sequence alignments of threepeptides derived from purified human 26S proteasome S5a with related regions within Arabidopsis MBP1 and yeast H9998.2. Their location withinthe MBP1 sequence is shown in A. Numbers refer to amino acid sequence position of MBP1. Residues that are identical or similar betweenArabidopsis MBP1 and the other proteins are displayed in black or gray, respectively.

similarity), rice (90% identity/98% similarity), and castor bean(91% identity/94% similarity), indicating that MBP1-typeproteins are highly conserved and widely distributed (Fig. 1A).The yeast sequence comprised the entire coding region,whereas the other sequences were derived from expressedsequence tags and thus incomplete. We confirmed the re-

ported sequence of the yeast clone by sequence analyses ofPCR products amplified from genomicDNA (data not shown).The derived amino acid sequence of the yeast gene exhibitssequence homology to Arabidopsis MBP1 throughout butterminates 110 amino acids earlier (Fig. 1A). Similarity was

especially evident at the N terminus and in and around the firsthydrophobic repeat of MBP1. Like MBP1, the extreme C-terminal region of the yeast protein is hydrophilic and enrichedin charged residues (Fig. 1A).The similarity in their apparent molecular masses and

affinities for multiubiquitin chains (ref. 13 and Fig. 2) sug-gested to us that Arabidopsis MBP1 and human S5a are

interspecific homologs. The similarity of MBP1 protein se-

quence to that derived from a human cDNA provided prece-

dence for a MBP1 homolog in humans (Fig. 1A). To explorethis possibility further, we compared the physical characteris-tics of MBP1 with S5a. Human 26S regulatory complex was

purified and the protein subunits were resolved by one- andtwo-dimensional gel electrophoresis. Of the 15 or so subunitsresolved by one-dimensional electrophoresis, S5a [which comi-grates with subunit S5b (ref. 11; Q.D., unpublished data)] hadan apparent molecular mass indistinguishable from MBP1(Fig. 34). As described, S5a was the only 26S proteasome

subunit that bound 1251-labeled multiubiquitin chains whentransferred to nitrocellulose (Fig. 3B and ref. 13). The bindingspecificity of S5a was similar to that of MBP1, having a highaffinity for chains containing four or more ubiquitins and littleor no affinity for ubiquitin monomers (Fig. 3C). The MBP1and S5a proteins were also indistinguishable when analyzed bytwo-dimensional gel electrophoresis. In the isoelectric focusingdimension, S5a was resolved from the more basic S5b subunit(Q.D., unpublished data) and could be easily identified basedon its ability to bind multiubiquitin chains (Fig. 4A and B). Inthis dimension, S5a and MBP1 had similar migration positions,indicating a common pI of -4.5 (Fig. 4A and D). Finally, S5awas immunospecifically recognized by MBP1 antisera (Fig.4C).Both their similar pl values and recognition by anti-MBP1

antisera provide compelling evidence that MBP1 and S5aproteins have related amino acid sequences as well as bindingaffinities. As a final confirmation that they are interspecifichomologs, we compared the predicted amino acid sequence ofMBP1 with those of peptides generated from S5a. Humanregulatory complex was resolved by one-dimensional gel elec-trophoresis, protein was transferred to nitrocellulose, and theregion of the blots containing S5a was treated with endopro-teinase Lys-C. Resulting peptides were subjected to N-terminal amino acid sequence analysis. Three peptides were

recovered that contained strong homology to the predictedpeptide sequence of Arabidopsis MBP1 (Fig. 1B). These pep-

tides were all located outside the region reported for thehuman cDNA sequence (Fig. 1A).

IVP1 61 I?PLZ3-SYeaSt l FHuman

CaenorhabditisRice E3 ~

MBP1 121 EjrAjE3 I 3R SYeaSt S. DEIRIRT3gN

299 DVNMSEAADEDQDLALALQMSMSGEESSEATGAGNNLLGNQAFISSVLSSaLPGVPNDPA

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FIG. 2. Expression and binding activity of recombinantArabidopsisMBP1. (A) Expression and purification of MBP1. Cell lysate from E.coli carrying pET25a (lane 1) or pET25a:MBPl (lane 2), or 5 ,ug ofpurified, recombinant MBP1 (lane 3) was fractionated by SDS/PAGEand stained for total protein. (B) Protein as in A was transferred tonitrocellulose membranes and probed with 125I-labeled multiubiquitinchains. Lane 3 contains 0.1 ,ug of purified, recombinant MBP1. (C)Protein as in A was transferred to nylon membranes and immunore-acted with antisera raised against hexahistidine-tagged MBP1. Lane 3contains 0.1 ,g of purified MBP1 and lane 4 contains 20 ,ug ofArabidopsis total leaf protein.

DISCUSSIONThe recognition and subsequent proteolysis of multiubiq-uitinated proteins is a pivotal step in a wide variety of cellularevents (1, 2). Given the pervasive expression pattern ofMBP1, evolutionary conservation among MBP1-like pro-teins across kingdoms, affinity for multiubiquitin chains, and

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FIG. 3. Comparison of physical properties and multiubiquitin chainbinding affinities of Arabidopsis MBP1 and human S5a. (A and B)Regulatory complexes from the human 26S proteasome (lane 1) orpurified MBP1 (lane 2) were electrophoresed on SDS gels, transferredto nitrocellulose, and stained with Ponceau S (A) or incubated with1251-labeled multiubiquitin chains (B). (C) 125I-labeled multiubiquitinchains before binding (lane 0) or after elution from S5a (lane 1) orMBP1 (lane 2). The majority of bound radioactivity was eluted fromeach immobilized protein. Exposures for lanes 0, 1, and 2 in C havebeen adjusted to account for total radioactivity. Molecular massmarkers are shown on the left. Migration positions of free, multi-ubiquitin chains are shown on the right. Hexahistidine-tagged MBP1gave similar results in these assays (data not shown).

homology to a known subunit of the 26S proteasome, it islikely that MBP1 serves an important role in the ubiquitinpathway by acting as a substrate recognition component ofthe 26S proteasome. The preferred association ofMBP1 withmultiubiquitin chains rather than single ubiquitins can ex-plain the requirement for multiubiquitination of target pro-teins prior to proteolysis by the 26S proteasome (10, 27) andthe ability of chain-termination mutants of ubiquitin (Lys-48to Arg) to dramatically impair ubiquitin-dependent prote-olysis (10).How MBP1 and homologs such as human S5a recognize

multiubiquitin chains is unclear. Because these proteins canD.: :t ..... OWssi.* .............. D ;. t* A .t

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proteasome or recombinant, purified ArabidopsisMBP1. Subunits of the regulatory complex were sepa-rated by two-dimensional gel electrophoresis andstained for total protein with Coomassie brilliant blue(A) or transferred to nitrocellulose and incubated with125I-labeled multiubiquitin chains (B), or with antisera

45 6 generated against MBP1 (C). The migration of molec-ular mass markers is shown on the left. (D) Purified

Coomassie MBP1 was resolved by two-dimensional gel electro-phoresis and stained for total protein. This protein wasexpressed as a hexahistidine-tagged version, purified,and cleaved with thrombin to remove the histidine tag.The more basic protein seen just to the right is likely asmall amount of the noncleaved protein. On the left ofeach two-dimensional gel, the 26S regulatory subunitswere separated by SDS/PAGE only (solid arrowheaddenotes the S5 band). Open arrows in A-C denote theS5a protein separated in two dimensions. In B and C,position of S5a was determined by Ponceau S staining.In D, open arrow shows the migration of purified MBP1.

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bind multiubiquitin chains even after denaturation andimmobilization on nitrocellulose, it suggests that the speci-ficity is derived from the primary sequence and/or a highlystable secondary structure(s). In the accompanying paperBeal et al. (28), using mutant ubiquitin molecules, implicatehydrophobic patches formed by repeating ubiquitin units inthe multiubiquitin chain-26S proteasome interaction. It istempting to speculate that the repeating hydrophobicpatches formed on multiubiquitin chains interact with aconserved, repeated hydrophobic domain (DXXLA-LALXXSM) that is found twice within the C-terminus ofMBP1. Such an interaction is consistent with our results, asthe MBP1 protein was originally isolated through its abilityto bind multiubiquitin chains under conditions expected tominimize ionic interactions (i.e., 0.5 M NaCl). Moreover,proteolytic mapping of S5a has implicated the C-terminalhalf, which is expected to contain the hydrophobic repeats,in chain recognition (data not shown). We predict thatthrough this interaction, MBP1 positions the multiubiquiti-nated target near other regulatory subunits within the 26Sproteasome involved in unfolding the target protein andtransporting the polypeptide chain into the lumen of the 20Sparticle where peptide bond cleavage occurs (29).The multiubiquitin chains used as a probe to isolate the

MBP1 cDNA and to assess the binding characteristics of MBP1and S5a were linked exclusively through Gly-76-Lys-48 (15,17). This is likely the predominant linkage found in vivo, asjudged by peptide mapping of purified multiubiquitin chains(17) and by the ability of Lys-48 -> Arg ubiquitin to effectivelyblock multiubiquitin chain formation when expressed in vivo(10). Multiubiquitin chains containing an alternative linkage(Gly-76 -* Lys-63) also exist in vivo and have been associatedwith the stress response in yeast (30, 31); recent data suggestthat S5a can bind alternative multiubiquitin linkages (32).Based on its binding properties, MBP1 should prove to be a

useful reagent for further studies of the ubiquitin system. Forexample, MBP1 can act as a potent inhibitor of ubiquitin-dependent proteolysis in vitro, presumably by interfering withtarget recognition by the 26S proteasome (33). Thus, ectopicexpression of wild-type or mutant derivatives of MBP1 or itshomologs may provide a novel and highly specific way toselectively inhibit the ubiquitin-dependent functions of thisprotease complex. Previous attempts to inhibit the 26S pro-teasome have relied on inactivating proteolytic activities of the20S core, which impacts both ubiquitin-dependent and ubiq-uitin-independent functions (34). The use of purified MBP1 asan affinity reagent may also facilitate the identification andisolation of ubiquitin pathway targets from crude cell extracts,which heretofore have been hampered by the low affinity ofubiquitin antibodies toward such conjugates. The identifica-tion of in vivo targets of ubiquitin conjugation will ultimatelybe essential to define the range of processes affected by thismodification.

Note Added in Proof. After completion of this work, Haracska andUdvardy (35) describe the cloning of a gene (,u54) encoding a 26Sproteaseome regulatory complex component of unknown functionfrom Drosophila melanogaster. This protein shares -45% amino acidsequence identity with MBP1 and likely represents a MBP1 homologin Drosophila.

We thank J. Walker for assistance in DNA sequencing, Dr. C.Pickart for multiubiquitin chains used in binding assays, and Dr. E.Vierling for theArabidopsis HSP70 andHSPI00 cDNAs. This workwasfunded by grants from the U.S. Department of Agriculture-NationalResearch Initiative Competitive Grants Program and Department ofEnergy/National Science Foundation/U.S. Department of Agricul-

ture Collaborative Program on Research in Plant Biology to S.v.N. andR.D.V. and by grants from the National Institutes of Health and theLucille P. Markey Charitable Trust to Q.D. and M.R.

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