Surface Hydrophobic Residues of Multiubiquitin Chains Essential for Proteolytic Targeting

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 861-866, January 1996 Biochemistry Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting RICHARD BEAL*t, QUINN DEVERAUXt, GANG XIA*t, MARTIN RECHSTEINERt, AND CECILE PICKART*t§ *Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, NY 14214; 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 Ubiquitin conjugation is a signal for degra- interaction between multiubiquitin chains and S5a remains dation of eukaryotic proteins by the 26S protease. Conjuga- completely uncharacterized. We used the recently determined tion of a homopolymeric multiubiquitin chain to a substrate structure of tetraubiquitin (Ub4) (19) as a starting point to lysine residue results in 10-fold faster degradation than does address this question by site-specific mutagenesis. Here we conjugation of monoubiquitin, but the molecular basis of report evidence concerning the molecular interactions respon- enhanced targeting by chains is unknown. We show that sible for enhanced proteolytic targeting by multiubiquitin ubiquitin residues L8, I44, and V70 are critical for targeting. chains. Mutation of pairs of these residues to alanine had little effect on attachment ofubiquitin to substrates but severely inhibited degradation of the resulting conjugates. The same mutations MATERIALS AND METHODS blocked the binding of chains to a specific subunit (S5a) ofthe Ubiquitin Mutagenesis, Expression, and Purification. Plas- regulatory complex of the 26S protease. The side chains mids encoding mutant ubiquitins were generated by whole- implicated in this binding-L8, 144, and V70-form repeating plasmid (circular) PCR using pPLhUb as template (20). This patches on the chain surface. Thus, hydrophobic interactions procedure employs back-to-back primers, one of which en- between these patches and S5a apparently contribute to codes the desired mutation (21). The presence of each muta- enhanced proteolytic targeting by multiubiquitin chains. tion was verified by determination of the complete coding sequence (fmol kit; Promega). Mutant genes under the control A major intracellular proteolytic pathway utilizes covalent of the A PL promoter in pPLhUb were expressed after heat conjugation of the conserved protein ubiquitin as a signal for induction of Escherichia coli strain AR58 (20). Briefly, 5-liter substrate recognition by a specific protease (1, 2). The ubiq- cultures were grown at 30°C to OD600 1.5 and then shifted uitin-mediated proteolytic pathway is the predominant mech- to 42°C for 2 h. The cells were collected by centrifugation and anism for turnover of short-lived proteins in eukaryotic cells lysed with a French press. The lysate was centrifuged at 10,000 (3). Substrates of this pathway include such critical regulatory x g. Perchloric acid (3.5%; vol/vol) was added to the super- proteins as the plant photoregulator phytochrome (4), c-Mos natant to precipitate nonubiquitin proteins. The acid super- (5), c-Jun (6), and NF-KB (7) as well as cyclins and other natant was neutralized and dialyzed, and ubiquitin was re- regulators of cell cycle progression (8-11). solved by cation-exchange chromatography (22). Ubiquitin was Ubiquitination occurs in three sequential enzymatic steps electrophoretically homogeneous after this step. (1): ATP-dependent formation of a thiol ester with ubiquitin- Assays of Degradation and Determination of Steady-State activating enzyme (El) at the ubiquitin C terminus (G76); Conjugate Level. Mutant ubiquitins were assayed for their ubiquitin transfer to a cysteine residue of a ubiquitin- ability to support the ubiquitination and degradation ofbovine conjugating enzyme (E2); and ubiquitin transfer to a lysine lactalbumin (Sigma) in ubiquitin-depleted rabbit reticulocyte residue of the substrate, catalyzed by a ubiquitin-protein ligase lysate or fraction II. Fraction II was prepared as described (23). (E3). Conjugated substrates are recognized and degraded by The standard incubation mixture contained (25-50 ,ll; 37°C) a multisubunit, ATP-dependent 26S protease that is assembled 50 mM Tris-HCl (pH 7.3), 5 mM MgCl2, 2 mM ATP, an from regulatory and catalytic (20S) complexes (12, 13). The ATP-regenerating system (10 mM phosphocreatine and 0.3 proteolytic cycle is completed upon regeneration of free unit of creatine phosphokinase per ml), 0.3 unit of inorganic ubiquitin by one or more specific isopeptidases (1, 14). pyrophosphatase per ml, - 1 mg of fraction II protein per ml, Substrates are most rapidly degraded when they are conju- and -2 x 105 cpm of 125I-labeled lactalbumin (106 cpm/,ug). gated to multiple molecules of ubiquitin (15, 16). A homopoly- Unless otherwise indicated, the concentration of ubiquitin was meric multiubiquitin chain, in which successive ubiquitins are 9 ,uM. This concentration is saturating for wild type (24) and linked by K48-G76 isopeptide bonds, is an especially potent was saturating for all mutant ubiquitins that were tested in this degradative signal: a substrate bearing a K48-linked chain of 8 regard (L8A, I44A, and L8A/144A). An aliquot was removed to 12 ubiquitins is degraded - 10 times more rapidly than a from the incubation mixture and quenched for SDS/PAGE substrate bearing a single ubiquitin at the same position (17). during the linear phase of the assay. Soluble counts were This length-dependent differential signal is reflected in the determined on a second aliquot after addition of trichloro- properties of subunit 5a (S5a) of the regulatory complex of the acetic acid (25). Ubiquintinated lactalbumin was visualized by 26S protease. S5a binds K48-linked multiubiquitin chains of n autoradiography of the dried gel; the region of the gel con- 2 4 with increasing affinity as a function of chain length but taining the conjugates was excised and counted in a y-counter has low affinity for monoubiquitin and chains of n c 3 (18). Conjugates bearing extended multiubiquitin chains presum- Abbreviations: El, ubiquitin-activating enzyme; E2, ubiquitin- ably undergo rapid degradation because they are efficiently conjugating enzyme; E3, ubiquitin-protein ligase; S5a, subunit 5a of targeted to the 26S protease via S5a (18). However, the 26S protease; Ub4, tetraubiquitin; Ub2, diubiquitin. tPresent address: Department of Biochemistry, School of Hygiene and Public Health, Johns Hopkins University, Baltimore, MD 21205. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" in Biochemistry, Johns Hopkins University School of Hygiene and accordance with 18 U.S.C. §1734 solely to indicate this fact. Public Health, 615 North Wolfe Street, Baltimore, MD 21205. 861 862 Biochemistry: Beal et aL Proc. Natl. Acad. Sci. USA 93 (1996) (25). Data were corrected by subtracting blanks derived from Chain Binding to S5a. Binding of radioiodinated chains and incubations lacking ubiquitin. Results are expressed relative to purified Ub4 molecules to S5a was assayed after fractionation the control with wild-type ubiquitin. of the 26S regulatory complex (20 ,ug per lane) by SDS/PAGE Inhibition of Degradation by Unanchored Ubiquitin (10% gel) and electrophoretic transfer of proteins to nitrocel- Chains. Degradation assays were carried out with 125I1 lulose (18). Chain concentration in the binding incubation lactalbumin as described above, except fraction II was depleted mixtures ranged from -0.2 to -0.6 jig/ml (see above). Bound of endogenous isopeptidase(s) by passage through ubiquitin- radioactivity was quantitated by Phosphorlmager analysis. Sepharose in buffer lacking ATP (26); the concentration of Binding data are expressed relative to a control of wild-type added wild-type monoubiquitin was 35 AM; and assays were ubiquitin chains in the same experiment. For P37C ubiquitin further supplemented with 1.8 ,uM ubiquitin aldehyde, an chains (and the corresponding wild-type control), incubations isopeptidase inhibitor (ref. 27; gift of Keith Wilkinson, Emory were done in the presence of 5 mM dithiothreitol, which University, Atlanta). Where indicated, unanchored chains slightly diminished the binding of wild-type chains (40% (distribution as in Fig. 3A) were added at a total concentration decrease) but largely eliminated an aberrantly high signal from of 0.8 mg of ubiquitin per ml. Without isopeptidase depletion P37C chains. This apparently reflected dissociation of disul- (see above), endogenous isopeptidase T completely disassem- fide-linked aggregates of the latter chains that remained bled added wild-type ubiquitin chains within minutes, and no partially competent in binding. These adducts could be visu- inhibition was seen (cf. Table 1). Isopeptidase T was purified alized by autoradiography after nonreducing SDS/PAGE of from bovine erythrocytes by a published procedure (26). E3/RAD6-Dependent Ubiquitination. Enzymes were puri- P37C ubiquitin chains. fied as described (25, 28). 125I-ubiquitin (2 AM; 8000 cpm/ pmol) was incubated with purified El (0.1 AM), purified yeast RESULTS RAD6(UBC2) (0.1 ,LM), and partially purified mammalian E3 Choice of Mutation Sites. The crystal structure of K48- in the presence of 0.2 mg of oxidized RNase per ml (Sigma) as of (Fig. substrate as described (25). In this

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