[Methods in Enzymology] Ubiquitin and Protein Degradation, Part B Volume 399 || Controlled Synthesis of Polyubiquitin Chains

[2] controlled polyubiquitin chain synthesis 21

[2] Controlled Synthesis of Polyubiquitin Chains

By CECILE M. PICKART and SHAHRI RAASI

Abstract

Many intracellular signaling processes depend on the modification ofproteins with polymers of the conserved 76‐residue protein ubiquitin. Theubiquitin units in such polyubiquitin chains are connected by isopeptidebonds between a specific lysine residue of one ubiquitin and the carboxylgroup of G76 of the next ubiquitin. Chains linked through K48‐G76 andK63‐G76 bonds are the best characterized, signaling proteasome degrada-tion and nonproteolytic outcomes, respectively. The molecular determi-nants of polyubiquitin chain recognition are under active investigation;both the chemical structure and the length of the chain can influencesignaling outcomes. In this article, we describe the protein reagents neces-sary to produce K48‐ and K63‐linked polyubiquitin chains and the use ofthese materials to produce milligram quantities of specific‐length chains forbiochemical and biophysical studies. The method involves reactions cata-lyzed by linkage‐specific conjugating factors, in which proximally anddistally blocked monoubiquitins (or chains) are joined to produce a partic-ular chain product in high yield. Individual chains are then deblocked andjoined in another round of reaction. Successive rounds of deblocking andsynthesis give rise to a chain of the desired length.

Introduction

Ubiquitin modifies a broad spectrum of proteins in eukaryotic cells (Penget al., 2003). All of these modified proteins share a common structuralfeature: at least one molecule of ubiquitin is linked through its C‐terminus(the carboxyl group of G76) to an amino group of the target protein. Usually,the linkage site is a lysine side chain of the target protein; less frequently, it isthe �‐amino group (Chen et al., 2004; Ciechanover and Ben‐Saadon, 2004).The covalently linked ubiquitin(s) modulates the stability, location, or func-tion of the target protein. Such regulation can follow directly from proteinmodification with a single ubiquitin, as in many instances of ubiquitin‐depen-dent endocytosis and trafficking (Hicke and Dunn, 2003). In other cases,appropriate functional regulation requires the initially conjugated ubiquitinto be extended into a polymer (a polyubiquitin chain) through the repeateduse of a lysine residue of ubiquitin as the conjugation site. Abundant evi-dence indicates that monoubiquitin and polyubiquitin can be functionallydistinct signals (Pickart and Fushman, 2004).

METHODS IN ENZYMOLOGY, VOL. 399 0076-6879/05 $35.00Copyright 2005, Elsevier Inc. All rights reserved. DOI: 10.1016/S0076-6879(05)99002-2

22 ubiquitin and ubiquitin derivatives [2]

Ubiquitin has seven lysine residues. Polyubiquitin chains linkedthrough K48 and K63 have well‐characterized and distinct roles; at leastsome other polyubiquitin chains probably serve novel signaling functions(Peng et al., 2003; Pickart and Fushman, 2004). Recent appreciation of thestructural and functional diversity of polyubiquitin chain signals, in con-junction with the discovery of multiple ubiquitin‐interacting domains, hasgenerated a demand for polyubiquitin chains in quantities necessary forbiochemical, structural, and biophysical studies. In this chapter we outlinemethods for the preparation of milligram amounts of K48‐ and K63‐linkedpolyubiquitin chains of defined length.

Functions of Polyubiquitin Chains

Ubiquitin’s best‐characterized function is to direct cellular proteins tothe 26S proteasome for degradation. The first studies of this role showed thatproteasomal targeting requires target protein modification with a polyubi-quitin chain linked through K48‐G76 isopeptide bonds (Chau et al., 1989).Because such chains are the principal proteasomal targeting signal andproteasomal proteolysis is an essential function, the K48R mutation inubiquitin is lethal in budding yeast (Finley et al., 1994). The extensive scopeof this function is further emphasized by the recent finding that ubiquitinitself is the most abundant ubiquitinated protein in budding yeast (Penget al., 2003). Although this mass spectrometric study used a nonquantitativemethod, the data suggested that K48 was the predominant linkage (Penget al., 2003). This conclusion is consistent with the failure of K‐to‐R muta-tions in ubiquitin, except K48R, to strongly alter the pattern or abundance ofubiquitinated proteins in the same species (Spence et al., 1995).

The Ub‐K63R mutation has no observable effect on proteasome func-tion. Instead, this mutation causes hypersensitivity to DNA damage(Spence et al., 1995). This phenotype reflects the modification of prolifer-ating cell nuclear antigen (PCNA) with a K63‐linked polyubiquitin chain,which in turn promotes a specific mode of DNA lesion bypass (Hoege et al.,2002). K63‐linked chains also act as signals in ribosomal translation, cer-tain endocytosis events, and (in mammals) kinase activation [reviewed inPickart and Fushman (2004) and Sun and Chen (2004)]. Although thereceptors that transduce K63‐chain signals remain to be identified in mostcases, it is unlikely that the proteasome is the destination of most substratesmodified by K63‐linked chains. A recent study detected all seven ubiqui-tin–ubiquitin isopeptide linkages in the budding yeast proteome (Penget al., 2003), and although definitive functional information is lacking forthe remaining linkages, some hints are available. Chains containing K29and K11 linkages have been implicated in the targeting of certain proteinsto proteasomes (Baboshina and Haas, 1996; Johnson et al., 1995). Chains


containing K6 and K27 linkages seem more likely be nonproteolytic signals(Morris and Solomon, 2004; Nishikawa et al., 2004; Okumura et al., 2004).

Polyubiquitin Chain Structure

At least two levels of structure are relevant when considering polyubi-quitin chains. The first is chemical structure; that is, which of ubiquitin’sseven lysines is/are used within the polymer? The significance of chemicalstructure has been rigorously analyzed for canonical K48‐linked chains.Here, in vitro analyses of the chain ligated to a substrate by its cognateE3 enzyme showed that only K48‐G76 bonds were present (Chau et al.,1989), whereas studies that use preassembled chains of defined structuredemonstrated the signaling competence of K48‐linked homopolymers(Thrower et al., 2000). Certain noncanonical chain signals are probably alsohomopolymers. The factors that catalyze K63‐chain synthesis in DNAdamage tolerance and kinase activation produce homopolymers in vitro(Deng et al., 2000; Hofmann and Pickart, 1999), and enzymes that produceK6‐ and K29‐linked homopolymers have been described (Nishikawa et al.,2004; Wu‐Baer et al., 2003; You and Pickart, 2001). Does a single chain evercontain more than one linkage? Proteomic evidence shows that the answercan be yes (Peng et al., 2003), but it is uncertain whether such chains arisepurposefully or adventitiously. Very little is known concerning the signalingproperties of heteropolymeric chains (Pickart and Fushman, 2004).

The second level of chain structure is conformational. Because ubi-quitin is a globular protein, chains with different chemical structuresmight possess distinctive ubiquitin–ubiquitin interfaces. Solution structuralstudies indicate that K48‐ and K63‐linked chains indeed have differentconformations in solution. At neutral pH, there are extensive ubiquitin–ubiquitin contacts in K48‐linked Ub2 (Varadan et al., 2002), whereas K63‐Ub2 adopts an extended conformation in which the covalent bond is theonly significant inter‐subunit contact (Varadan et al., 2004).

Enzymes Used for In Vitro Chain Synthesis

Because polyubiquitin chains are assembled through isopeptide (versuspeptide) bonds, enzymatic synthesis is necessary. Therefore, one’s ability tomake a given chain presupposes the availability of a conjugating enzyme(s)that is (1) linkage‐specific and (2) displays robust activity toward free ubi-quitin. So far, we have identified such factors for K48‐ and K63‐linked chains.Although a number of other linkage‐specific factors have been reported (seepreceding), the activity of most of these factors toward free ubiquitin has notbeen carefully investigated. Consequently the suitability of these enzymesfor the method outlined in the following remains uncertain.


The conjugating enzyme should be highly linkage‐specific. This issue isoften addressed using mutant forms of ubiquitin (see Chapter 1). However,mass spectrometry provides the most rigorous criterion of linkage speci-ficity (Peng et al., 2003; Pickart and Fushman, 2004). Application of asemiquantitative version of this method to K48‐ and K63‐linked chainssynthesized by the methods described in the following has shown thatonly the desired linkage is detectable (J. Peng and C. Pickart, unpublisheddata). In contrast, this criterion was not met when we used a specific E3enzyme to synthesize K29‐linked chains (M. Wang, J. Peng, and C. Pickart,unpublished data).

For the synthesis of K48‐linked chains, we use the mammalian enzymeE2‐25K (Chen et al., 1991). The biological function of E2‐25K is uncertain,but its activity is well characterized in the in vitro setting. Of particularimportance, although E2‐25K binds free ubiquitin weakly, it is very activeat the high concentrations of ubiquitin used in chain synthesis (Haldemanet al., 1997). The same is true of the yeast Mms2/Ubc13 complex (a UEV/E2 heterodimer), which participates in K63‐chain synthesis in DNA dam-age tolerance in vivo (Hofmann and Pickart, 2001). These conjugatingfactors are conveniently expressed in Escherichia coli.

Polyubiquitin Chain Recognition

The past several years have seen the discovery of a small group of proteindomains that bind ubiquitin and/or polyubiquitin chains, including the ubi-quitin interacting motif (UIM), ubiquitin‐associated domain (UBA), NPL4zinc finger (NZF), and CUE domain (coupling of ubiquitin to endoplasmicreticulum degradation) [see Buchberger (2002); Hicke and Dunn (2003);and Pickart and Fushman (2004)]. Each domain occurs in a modest numberof proteins in a given species. If polyubiquitin chains with different struc-tures represent unique signals, there should be downstream binding factorsthat can discriminate among different chains. Recent studies identified aUBA domain and a zinc finger domain that preferentially bind K48‐ andK63‐linked chains, respectively (Kanayama et al., 2004; Raasi et al., 2004).The ability to generate large quantities of structurally defined polyubiquitinchains will aid in the discovery of new linkage‐specific binding proteins andshould facilitate structural biology aimed at explaining the molecular basisof such linkage specificity. Enabling such studies has been an importantmotivation for developing the methods described in the following.

A Method for Controlled Synthesis of Polyubiquitin Chains

The method is outlined in Fig. 1 (Piotrowski et al., 1997). It involves aseries of enzymatic reactions catalyzed by the linkage‐specific enzymes

FIG. 1. Synthesis of K48‐linked Ub4. This scheme outlines the steps in the synthesis of

K48‐Ub4 (see the text). The circles denote Ub molecules; the shading lets the reader

keep track of the different ubiquitins in the chain. In the doubly blocked Ub4 molecule, D77

of Ub‐1 is the proximal chain terminus and C48 of Ub‐4 is the distal terminus.


discussed previously. We refer to the end of the chain that would normallycarry unconjugated G76 as the proximal end, whereas the end that wouldnormally carry unconjugated K48 is the distal end (see Fig. 1). In eachround of reaction, proximally and distally blocked monoubiquitins (orchains) are joined to produce a doubly blocked chain. The proximal blockconsists of an extra C‐terminal residue (D77) that is labile to ubiquitincarboxyl–terminal hydrolases (UCHs). The distal block consists of a cyste-ine residue, placed at the normal conjugation site, that is later converted toa lysine mimic (S‐aminoethylcysteine) through alkylation. Successiverounds of deblocking and conjugation give rise to a chain of any desiredlength. The method is presented in detail for K48‐linked chains and inoutline form for K63‐linked chains. All of the plasmids mentioned in thischapter are available to academic researchers on request. When proteinreagents are commercially available, we mention current suppliers.


Expression and Purification of Proximally and Distally BlockedUbiquitin Monomers

Ub2 linked through K48 or K63 is synthes ized from a mat ched pair ofmonom eric ubiq uitin reactants . One monom er, which cann ot conjuga tethrough its C ‐ terminus , carries an extra resi due (D77 ). The other monom ercanno t conjuga te through its lysine, be cause it carries a muta tion to cyst e-ine (or argi nine) at this pos ition. The blocked ubiqu itins are produ ced inE. coli . We use pET3a plasmi ds that speci fy untagged ubiqu itins, whi chyield 50– 100 mg of purified ubiqui tin per liter of cultu re. The pro ceduresbelow are for a 2 ‐liter scale ex pression. To scale up, all volumes should beincrea sed in a direc tly proportio nal manne r. Tag ged version of ubiqu itincan also be used to synthes ize K48‐ link ed chains.

Protocol for Ubiquitin Expressio n

1. Two days before expression, stre ak E. co li cells carry ing theappro priate plasm id on sele ctive medi um. We express the ampi cillin ‐marke d ubiqui tin plasm ids (pET3a ) in the BL21( DE3) strain carry ing achlor ampheni col‐ marke d he lper plasm id, pJY2, that specifies the AGA ‐decod ing arginine tRNA , as well as T7 lysozy me (LysS) ( You et al., 1999 ).Alterna tely, one can us e comm ercial E. coli strains that ov erexpre ss raretRNA s and carry the LysS gen e. Bec ause the synthet ic genes that form thebasis of most ubiquitin cDNAs are rich in AGA codons (this codon is alsoused for lysine‐to‐arginine mutations), failure to supply additional tRNAcan result in misincorporation of lysine for arginine at AGA codons [seeYou et al . (1999) ]. The presenc e of LysS blocks prema ture ubiqu itinexpression that could otherwise be toxic (You et al., 1999).

2. To make the starter culture, inoculate 25 ml of 2 � YT medium(containing appropriate antibiotics) with a single colony. For pET3aplasmids, the starter culture should not reach saturation to avoid lossof cell viability. It is convenient to start this culture in the morning1 day before large‐scale expression, growing at 37� to OD600 � 0.6(requires 5–8 h). This starter culture is refrigerated overnight.

3. Dilute 20 ml of starter culture into 2 liters of medium. Culture withgood aeration at 37� to OD600 � 0.6, then induce with 0.4 mM IPTG.Culture for 4 h more, then harvest cells and freeze at �80�. Cell pellets canbe stored indefinitely.

Protocol for Ubiquitin Purification

The following procedure describes a simple purification of untaggedubiquitin that takes advantage of ubiquitin’s solubility in perchloric acid.Although the protein is >90% pure after the acid precipitation step

FIG. 2. Ubiquitin and chain synthesis/purification (Coomassie‐stained gels). (A) Ubiquitin

expression. Proportional aliquots of stages in the purification of Ub‐K29R,D77 were analyzed.

WCE, whole‐cell extract; SE, extract after centrifugation; PCA sup, supernatant of perchloric

acid precipitation. (B) Synthesis of K48‐linked chains. Lanes 1 and 2, 48‐mg scale synthesis of

K48‐Ub2 at time zero and 4h, respectively. Lanes 3 and 4, 20‐mg scale synthesis of K48‐Ub4at time zero and 2h, respectively. Asterisk denotes a small amount of Ub6 that was probably

formed after carboxypeptidase‐catalyzed removal of D77 from the distally deblocked dimer.

(C) Purification of K48‐Ub2. The 50‐mg scale synthetic reaction was applied to a 6‐ml Resource

S column (Amersham‐Pharmacia Biotech) preequilibrated with 50mM ammonium acetate, pH

4.5. The column was eluted with a 60‐ml gradient of NaCl (0–0.6M). Fractions (40 � 1.5ml)

were collected; 2�l aliquots of the indicated fractions were analyzed. The bar highlights the

fractions that were pooled and concentrated. A total of 44mg of Ub2 was recovered (88%).


(Fig. 2A, lane 5), gradient cation exchange chromatography will removeUV‐absorbing contaminants that would otherwise prevent the use of UVabsorbance to determine protein concentrations.


1. Prepare 50 ml of lysis buffer consisting of 50 mM Tris‐HCl (pH7.6), 1 mM phenylmethylsulfonyl fluoride, 50 �M tosyllysylchloromethylketone, 2.5 �g/ml leupeptin, 5 �g/ml soybean trypsin inhibitor, 0.02% (v/v)NP‐40, and 0.4 mg/ml lysozyme. If the ubiquitin contains a cysteineresidue, include 1 mM DTT in the lysis buffer. (Protease inhibitors anddetergent can be purchased from Sigma or another biochemical supplycompany.)

2. Add the buffer to the frozen cell pellet. To lyse, pipet up and downgently with a plastic transfer pipet. As the cells lyse, the suspension willbecome viscous from released DNA.

3. Digest the DNA by adding MgCl2 to 10 mM and DNAseI (Sigma)to 20 �g/ml. Pipet up and down (or place tube on a rocker) until the DNAis lysed as judged by normal viscosity; this usually requires 10–20 min atroom temperature.

4. Centrifuge the lysate at 8000 rpm for 20 min in a Sorvall SS‐34rotor. Carefully transfer the supernatant to a chilled beaker with a smallstir bar (on ice).

5. Slowly add 0.35 ml of 70% perchloric acid while stirring vigorously.The solution will immediately turn milky. Continue stirring for 10 minafter all the acid has been added.

6. Centrifuge the suspension as in step 4.7. Transfer the supernatant to dialysis tubing with a molecular weight

cutoff of 3.5 kDa. It is simplest to use extra‐wide tubing.8. Dialyze the perchloric acid supernatant in the cold against 2 liters

of 50 mM ammonium acetate (pH 4.5) for at least 4 h. Repeat with 2 litersof fresh buffer. If the ubiquitin contains a cysteine residue, include 1 mMEDTA and 1 mM DTT in the buffer.

9. While the protein is dialyzing, pour a 15‐mL; column of S‐Sepharose Fast Flow (Amersham‐Pharmacia Biotech) and equilibrate itwith 10 column volumes of 50 mM ammonium acetate, pH 4.5 (plus 1 mMeach of EDTA and DTT if the ubiquitin contains a cysteine).

10. Apply the dialysate to the column. All of the ubiquitin should bind.Wash the loaded column with 1 volume of equilibration buffer, and thenelute at 2–3 ml/min with a 300‐ml linear gradient of 0–0.5 M NaCl in thesame buffer, collecting fractions of 4 ml. (Note: one can do this step with anopen column and a peristaltic pump or with an automated chromatographydevice.) Locate the peak by SDS‐PAGE/Coomassie staining of fractionaliquots or by UV absorbance. Ubiquitin elutes at �0.24 M NaCl. Pool thepeak fractions and concentrate in a centrifugal concentrator (we use 15 mlAmicon Ultra devices with a 5‐kDa mass cutoff).

11. Exchange the ubiquitin into a buffer compatible with subsequentenzymatic reactions. Tris‐HCl (pH 7.6), 10 mM, is convenient. If there is a


cysteine residu e, include EDTA (0.5 m M ) and DTT (1 m M ) in this buf fer.Buffer exchan ge can be done by diluti ng an d recon centratin g severa l times.To minimiz e protein losses during freezing and thawing, concent rate theubiquiti n to at leas t 50 mg/ml (all of the ubiqui tin mutants used here aresoluble to at least 110 mg/ml). The concent ration of ubiqui tin is determi nedby UV absorban ce. A 1 mg/ml solution of ubiqui tin has OD280 ¼ 0.16.Concen trated stocks of ubiq uitin can be store d indefini tely at � 20 � .

Expression and Purification of Conjugating Enzymes

For K48 ‐ chain synthes is catalyze d by E2‐ 25K, we use untagged recom -binant enzym e purified by con vention al ch romatograph y ( Haldema n et al.,1997 ). How ever, most resear chers will find it easi er to pr oduce a GST ‐tagged version of E2‐ 25K using pGE X*E225K ( Haldema n et al ., 1997 ). Inthis case, release bound E2 ‐25K from GSH beads (S igma) using thrombin.Use 1 U throm bin (Am ersham ‐ Pha rmacia Bio tech) per 100 �g of fusi onprotein, incubat ing for severa l hours at room tem peratur e. Cleavage isrecommende d, becau se GST ‐ fused E2‐ 25K is a less robust con jugatingenzyme than free E2 ‐ 25K. This E2 (also called UbcH1) is comm erciallyavailable from Bosto nBiochem and Biomol. It is only active with mam ma-lian E1 e nzymes, which are comm ercially availabl e from the same suppli-ers. One cautionary note: on occasion, commercial E1 preparations havebeen found to be contaminated with deubiuqitinating activity (M. Petroskiand R. Deshaies, personal communication, 2004). The interested readershould consul t the chapter by Beaude non a nd Hui bregtse (2005) andcolleagues for information about recombinant E1 enzymes.

For K63‐chain synthesis catalyzed by the Mms2/Ubc13 complex, we usethe budding yeast versions of these proteins. We use mammalian E1, butyeast E1 can be substituted. Ubc13 is expressed as a GST fusion andreleased from GSH beads by thrombin cleavage (Hofmann and Pickart,2001). PolyHis‐tagged Mms2 is expressed using pET16b‐Mms2; it isprimarily insoluble. We purify the protein under mild denaturingconditions (4 M urea) using nickel beads (Novagen) and renature it bydialysis (VanDemark et al., 2001). The human Mms2/Ubc13 complex iscommercially available from BostonBiochem.

Synthesis of K48‐Linked Chains

Protocol for Synthesis and Purification of K48‐Ub2

The experienced protein chemist can also consult a more concise ver-sion of the protocol in the following (Raasi and Pickart, 2005).


1. Prepare PBDM8 buffer containing 250 mM Tris‐HCl (50% base, pH8.0), 25 mM MgCl2, 50 mM creatine phosphate (Sigma P7396), 3 U/mlinorganic pyrophosphatase (Sigma I1891), and 3 U/ml creatine phosphoki-nase (Sigma C3755). Also prepare a neutral 0.1 M ATP solution (SigmaA2383). Both solutions are stable at �20�.

2. To synthesize K48‐Ub2, combine Ub‐D77 and Ub‐K48C at 7.5mg/ml each in an incubation containing one‐fifth volume of PBDM8 plus2.5 mM ATP, 0.5 mM DTT, and 20 �M E2‐25K. Avoid higher concentra-tions of DTT, which can trap ubiquitin by reacting with the E2‐ubiquitinthiol ester. Remove 1 �l for SDS‐PAGE analysis. (Note: do not boil poly-ubiquitin chains, because this can cause nonspecific cross‐linking to yieldproducts that resemble chains.) The conjugation reaction, initiated byadding 0.1 mM mammalian E1, requires 4 h at 37� (lane 2, Fig. 2B). Toavoid having unreacted ubiquitin monomer, use precisely equal concen-trations of Ub‐K48C and Ub‐D77. At the end of the incubation, add DTT(5 mM, freshly prepared) and EDTA (1 mM), then incubate at roomtemperature for 20 min to reduce disulfide‐linked chains that couldprecipitate later. Remove 1 �l for SDS‐PAGE.

3. Add one‐fifth volume of 2 N acetic acid to the reduced reaction; checkthat the pH is�4 by spotting 1 �l onto pH paper. Apply the acidifiedmixtureto a cation exchange column preequilibrated with buffer A (50 mM ammo-nium acetate, pH 4.5, 1 mM EDTA, 5 mM DTT). Recommended mediaincludeMono S, Resource S, or S‐Sepharose Fast Flow (all fromAmersham‐Pharmacia Biotech), using 1‐ml beads per 20 mg of total ubiquitin. Wash theloadedcolumnwith�3volumesofBufferA (save toverify thatUb2 is absent).Elute the column with a linear gradient of NaCl (0–0.6M) in Buffer A, using10–40 column volumes and collecting 40–60 fractions. Ub2 elutes at �0.33MNaCl. The fractions should be examined by SDS‐PAGE to reject those thatcontain significant Ub1 or Ub3 (Fig. 2C); often, a small amount Ub3 is formedduring the synthetic reaction because of carboxypeptidase‐catalyzed removalof D77 from Ub‐D77 (a low level of carboxypeptidase activity contaminatessome preparations of E2‐25K). Good resolution of Ub2 from Ub1 and Ub3(Fig. 2C) is best obtained with an automated chromatography device.Pool and concentrate thepeak fractions ofUb2, thenexchange the sample intostorage buffer (20 mM Tris‐HCl, pH 7.6, 0.5 mM EDTA, 2 mM DTT). Toreduce losses caused by nonspecific absorption, concentrate to 30–80 mg/mlbefore storing at�80�.WeuseAmiconUltra 4‐ml concentratorswith a 5‐kDamass cutoff. Polyubiquitin chains tend to precipitate when left at pH 4.5.Therefore, we recommend pooling, concentrating, and exchanging intostorage buffer on the day that the column is run. Should precipitation occur,the chains canbedissolved inabuffer of 10 mMTris (pH7.6), 1 mMEDTA,5mM DTT, and 8M urea. Remove the urea by dialysis.


Protocols for Deblocking Reactions

The Ub2 resulting from the procedures described in the preceding isdoubly blocked; it carries D77 at its proximal terminus and a C48 residue atits distal terminus (Fig. 1). To generate Ub4, half of the Ub2 is deblocked atits proximal terminus, exposing G76, whereas the remainder of the Ub2 isdeblocked at its distal terminus, by alkylating to introduce a lysine mimic.The two singly blocked dimers are later conjugated to produce Ub4.

1. D77 is removed enzymatically by treating Ub2 with ubiquitin C‐terminal hydrolase (UCH). We use yeast ubiquitin hydrolase‐1 (YUH1),which we express and purify according to published procedures (Johnstonet al., 1999), but commercially available UCH enzymes can be substituted(available from BostonBiochem or Biomol). To the doubly blocked Ub2(30–80 mg/ml), add: 50 mM Tris‐HCl (pH 7.6), 1 mM EDTA, and 1 mMfresh DTT. Initiate deblocking by adding purified UCH to a finalconcentration of 16 �g/ml. Quantitative removal of D77 occurs in 60 minat 37�. Add 4 mMmore DTT and incubate for 10 min at room temperature(to reduce any disulfide bonds). Remove the UCH by passing the reactionmixture through a 0.5‐ml Q‐Sepharose Fast Flow column preequilibratedwith Q buffer (50 mM Tris‐HCl, pH 7.6, 1 mM EDTA, 5 mM DTT).Collect the unbound fraction together with four washes (0.5 ml Q buffereach), reconcentrate to the original volume, and determine the concentra-tion of the proximally deblocked dimer by UV absorbance.

2. To alkylate the remaining doubly blocked dimer (30–80 mg/ml) add0.2 M Tris‐HCl, pH 8.0, and 1 mM EDTA. Initiate alkylation by addingethyleneimine to 55 mM (available from Chemservice). The reactionrequires 60 min at 37�. Ethyleneimine must be removed to prevent sub-sequent inactivation of E1 and E2‐25K. One can dialyze the incubationagainst 1 liter of 10 mM Tris‐HCl, pH 8.0 (overnight at 5�). Or one canrepeatedly concentrate and dilute with 10 mMTris‐HCl, pH 8.0, 2 mMDTTin a centrifugal concentrator until [DTT] ¼ [ethyleneimine]. Concentratethe distally deblocked dimer to 30–80 mg/ml before freezing. Ethylene-imine is toxic and should be handled with care. Vials should be opened onlyin a fume hood, and manipulations involving the concentrated stock shouldbe performed there as well. Unused ethyleneimine can be diluted into 10–50volumes of alkaline DTT and allowed to sit for 24 h before disposal.

Protocol for Synthesis and Purification of K48‐Ub4

Conditions are the same as in the synthesis of K48‐Ub2 (above), exceptas follows. First, the reactants are the proximally and distally deblockedUb2 molecules (above). Second, each reactant is added at 10 mg/ml. Finally,


the incubation can be shortened to 2 h (Fig. 2B, lanes 3 and 4). Thepurification method is the same as for Ub2 (above), except that Ub4 bindsmore tightly to the cation exchange column, so a gradient of 0–0.7 M NaClis used.

Synthesis of K48‐Linked Chains of Other Lengths

The principles are as described previously. Because resolution duringcation exchange purification is only possible if the chains differ significantlyin their lengths, long chains should be made by joining two chains of similarlengths. For example, make Ub12 by linking Ub6 to Ub6 (or Ub4 to Ub8)rather than by linking Ub2 to Ub10. Longer chains require higher saltconcentrations to elute, so gradients should be adjusted accordingly. Wehave successfully made chains up to n ¼ 12 (Raasi and Pickart, 2005; Raasiet al., 2004; Thrower et al., 2000). Once the chain has reached its finallength, the distal C48 residue can be alkylated with ethyleneimine (above)or iodoacetamide, if desired, to reduce the potential for precipitation.We add iodoacetamide �threefold excess over total thiol and incubatefor 1–2 h at 37� (pH 7.6). Excess iodoacetamide should be removed byrepeated concentration/dilution or dialysis.

Synthesis of K63‐Linked Chains

The principles and procedures are similar to those outlined for K48‐linked chains, with several differences. First, the synthetic reaction contains8 �M each of yeast Mms2 and Ubc13 in place of E2‐25K. Second, thebuffer is PBDM7.6. (PBDM7.6 is the same as PBDM8, except that (1) Tris,pH 7.6 (24% base), is substituted for Tris, pH 8, and (2) 10 mM ATP isincluded in PBDM7.6. Accordingly, it is not necessary to add ATP inde-pendently to the conjugation reaction). Third, yeast or mammalian E1 canbe used. Fourth, tagged ubiquitins should not be used with the yeast Mms2/Ubc13 complex, because they are inefficiently conjugated (Hofmann andPickart, 2001). Finally, this complex discriminates against alkylated ubiqui-tin, making it impractical to deblock the distal terminus with ethyleneimine(Hofmann and Pickart, 2001). Therefore, we build up K63‐linked chainsone ubiquitin at a time. K63‐Ub2 can be synthesized from Ub‐K63R andUb‐D77 at 10 mg/ml each (use of Ub‐K63R instead of Ub‐K63C eliminatesprecipitation problems), purified, and deblocked with UCH according tothe same procedures used for K48‐Ub2. Then conjugate the proximallydeblocked Ub2 to Ub‐D77, yielding K63‐Ub3. After purification and de-blocking, K63‐Ub3 is conjugated to Ub‐D77 to yield K63‐Ub4 (Hofmannand Pickart, 2001).


Factors Contributing to Yield and Recovery

In our original procedures, we removed the conjugating enzymesby subtractive anion exchange before acidifying the reaction mixture(Hofmann and Pickart, 2001; Piotrowski et al., 1997; Raasi and Pickart,2005). This step is dispensable for chains up to n ¼ 4 but may be necessaryto ensure maximum purity of longer chains. In general, good normalizationof the molar concentrations of the chain reactants will maximize conver-sion, simplify purification, and optimize the yield. However, when adding asingle ubiquitin to a preexisting chain, use a 10% stoichiometric excess ofthe monomer reactant to force complete conversion of the chain to then þ 1 product; this will improve resolution during cation exchange. Forthe purification scheme discussed previously, we usually recover 70–90% ofthe input ubiquitins in the specific chain product in 30‐ to 60‐mg scalereactions. Recovery during UCH‐dependent deblocking is 80–90%, where-as recovery during ethyleneimine alkylation is 75–90%; both recoveriesare better in large‐scale reactions. Ubiquitin and polyubiquitin chainsare rather sticky; nonspecific absorption to surfaces significantly reducesrecovery. Reusing columns, avoiding glass tubes, and maximizing proteinconcentrations will counteract this problem.

Specialized Applications

It is straightforward to modify the preceding protocols for selectedpurposes. For example, one can make structurally defined heteropolymers.We used the Mms2/Ubc13 complex to conjugate a preassembled K48‐chain to Ub‐K63 in a ubiquitin‐dihydrofolate reductase fusion protein(M. Ajua‐Alemanji and C. Pickart, unpublished data). Here we synthesizedthe K48‐linked chain from monomers carrying the K63R mutation to avertchain–chain conjugation in the final step. There are other instances inwhich the appropriate use of additional mutations can be used to avoidself‐ligation of chains by other conjugation factors. Thus, to conjugatepreassembled K63‐Ub4 to an internal lysine residue of a model substrateusing an E3, we synthesized the chain from monomers carrying the K48Rmutation to block self‐ligation of the chain in the E3‐catalyzed step (P.‐Y.Wu and C. Pickart, unpublished data).

Any doubly blocked, K48‐linked chain (Fig. 1) carries a unique cysteineat its distal terminus that can be used to introduce a thiol‐reactive cross‐linker, spin label, or fluorescent group (Fig. 3B). Alternately, such agroup can be introduced at the proximal end by reacting the distal cysteine(if present) with iodoacetamide and then conjugating the chain to Ub‐G76C (Fig. 3C). UCH is included in the latter reaction to remove the

FIG. 3. Specialized chains: representative examples. (A) Chimeric mutant chains. In this

example, the Ub‐K48C that was used to make the distally deblocked dimer on the right

carried an additional mutation (indicated by the shading and the letter ‘‘M’’). (B) Distally

modified chains. The C48 thiol group at the distal terminus of this doubly blocked tetramer

can be modified in a site‐specific manner (left to right) by disulfide exchange or reaction with

iodoacetyl or maleimide derivatives. (C) Proximally modified chains. C48 of the doubly

blocked tetramer from (A) is reacted with iodoacetamide, followed by another round of

conjugation to introduce Ub‐G76C at the proximal terminus. The C76 thiol group can then be

modified as shown in (B).


chain’s or iginal proximal block; the G76C muta tion creates a new proximalblock. Finally, one can intr oduce a muta nt ubiqu itin at any point in thechain by incor porating addit ional mutatio ns into the singly blocked ubiqui -tins ( Fig. 3A ). For example, to test the im portanc e of Ub ‐ L8 in K48 ‐ chainbindi ng to proteas omes and UBA domai ns, we made a series of chimeri ctetrame rs in which the L8A muta tion was intr oduced into pairs of mono-mer units in all possible combi nation s (Raas i et al ., 2004; Thr ower et al .,2000 ). We also synthes ized a K48 ‐ Ub4 whose proxim al ubiquiti n carriedthe P37C muta tion with or without the L8A muta tion. We introduced across ‐ linker a t C37 and tested how the L8A muta tion affe cted cross ‐li nkingof the chain to pro teasomes (Lam et al., 2002 ). The av ailable spectru m ofsuch ‘‘d esigner chains’’ is limi ted principal ly by the effor t involved inclonin g an d ex pressing the ne cessary ubiqui tin muta nts.

Acknowledgments

We thank Matt Steele for providing the data shown in Fig. 2A and members of the

Pickart laboratory for comments on the manuscript. Our research on polyubiquitin chains is

funded by grants from the NIH.


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