THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 22, Issue of June 3, pp. 15710-15717,1994 Printed in U.S.A.

Characterization of Denatured Protein Inducers of the Heat Shock (Stress) Response in Xenopus Zaeuis Oocytes*

(Received for publication, March 4, 1994)

Laura C. Mifflinl and Robert E. Coheni;§fl From the $Department of Chemistry and Biochemistry and The Molecular Biology Institute, University of California, Los Angeles, California 90024 and the $Department of Biochemistry, University of Zowa, Iowa City, Iowa 52242

In addition to thermal stress, a large variety of physi- cal and chemical treatments are known to induce heat shock gene expression. Denatured protein, thought to result from the stress condition, has been postulated to act as the common signal. Accordingly, of three pairs of native and denatured proteins injected into Xenopus laevis oocytes, only the denatured derivatives induced expression of a reporter gene from a heat shock pro- moter (Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986) Science 232, 522525). These observations are ex- tended here. Protein denaturation per se is shown to be insufficient for heat shock induction; although reduced and carboxymethylated bovine serum albumin (rcm- BSA) and a-crystallin elicited a stress response, many other denatured proteins had no effect. Methylation of protein lysines, done to prevent ubiquitination, sup- pressed heat shock induction by rcm-BSA, but enhanced induction by a-crystallin. Thus, the potential for a pro- tein to be ubiquitinated is independent of its ability to induce the stress response. Instead, aggregation distin- guished the proteins that were effective stress inducers, and the formation of large aggregates correlated with the magnitude of the response. This correlation may de- rive in part from decreased in vivo degradation rates of the inducer proteins. An apparent requirement for stress response induction that the inducer proteins be injected directly into the oocyte nucleus may relate to this issue of in vivo stability. The dependence of the stress response on the amount of injected protein is non- linear and of a form consistent with the titration of a factor that otherwise suppresses heat shock gene expression.

The response by cells to heat involves the shut-down of nor- mal transcription and translation in favor of the enhanced pro- duction of a small number of specific proteins, called heat shock proteins (hsp’; for reviews, see Lindquist and Craig (19881, Schlesinger (19901, and Hightower (1991)). Cells subjected to a wide variety of stress conditions in addition to heat, such as

Service Grant R 0 1 GM37666 (to R. E. C.) and a fellowship from United * This work was supported in part by United States Public Health

States Public Health Service Training Grant GM07185 (to L. C. M.).

Proteins & The Heat Shock Response, Cold Spring Harbor Laboratory Preliminary results have been reported in abstract form ((1991) Stress

Press, Cold Spring Harbor, NY). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisenent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence and reprint requests should be addressed:

Tel.: 319-335-8545; Fax: 319-335-9570. Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242-1109.

The abbreviations used are: hsp, heat shock protein; BSA, bovine

N-methylated; cbm, carbamoylated; rca, reduced and carboxamido- serum albumin; rcm, reduced and carboxymethylated; me, reductively

methylated I-BSA, iodinated BSA, DLS, dynamic light scattering.

ethanol, oxidizing agents, viral infection, heavy metals, and amino acid analogues, produce the so-called heat shock or stress response (Nover, 1984). Extensive protein aggregation in stressed cells, particularly in the nucleus and nucleolus, also is observed to accompany a stress (e.g. Pelham (19841, Nover et al. (1989), Dubois et al. (1991), and Beckmann et al. (1992)). The realization that protein denaturation may be a common conse- quence of various stress conditions has led to the idea that denatured proteins present some feature recognized for initia- tion of a stress response (Hightower, 1980; Goff and Goldberg, 1985; Ananthan et al., 1986). Specific denatured proteins i n vivo can elicit a stress response. An unfolded, mutant form of A repressor induced a stress response in Escherichia coli (Parsell and Sauer, 19891, whereas its folded counterpart had no effect. In Drosophila, tissue-specific constitutive expression of heat shock proteins correlated with the expression of mutant forms of actin (Hiromi et al., 1986; Okamoto et al., 1986). Ananthan et al. (1986) reported that the stress response could be elicited by the direct introduction via microinjection of denatured proteins intoxenopus laevis oocytes, whereas the native proteins had no effect.

The mechanism whereby denatured proteins can elicit a heat shock response is unclear, although candidates for the machin- ery that monitors levels of denatured proteins are known. Members of the 70-kDa heat shock protein family frequently are cited in this context (e.g. Craig and Gross (1991) and ref- erences therein). Such a role is suggested by the several known chaperone activities of hsp70-like proteins combined with evi- dence for autoregulation of hsp7O expression; this is elaborated under “Discussion” and in the accompanying paper (Mifflin and Cohen, 1994). Another stress sensor could be the system re- sponsible for the intracellular degradation of abnormal and denatured proteins; in eukaryotes, the ubiquitin-dependent degradation system functions in this capacity (Ciechanover et al., 1984; Finley et al., 1984). Several observations have linked the ubiquitin system to the stress response. Ubiquitin itself is a stress protein (Bond and Schlesinger, 19851, as are some enzymes of the ubiquitin-mediated protein degradation system. Deletions of the polyubiquitin gene in yeast and mutational alterations in some ubiquitin pathway enzymes renders cells hypersensitive to stress (Finley et al., 1984; Finley et al., 1987; Seufert and Jentsch, 1990). Presumably, the ubiquitin system is employed to remove the increased load of denatured protein that arises from a stress condition. Although the level of ubiq- uitin itself does not appear to regulate stress response induc- tion (Bond et al., 1988; Lis and Lee, 19881, it is possible that conjugates of ubiquitin with denatured protein are sensed for stress response regulation.

Properties of denatured proteins that are critical to stress response induction can be studied by the use ofX. laevis oocytes to assay heat shock specific gene expression. Although Xenopus oocytes do not respond to stress by transcription of their own heat shock genes (King and Davis, 19871, they can activate

15710

Denatured Protein Inducers of the Stress Response 15711

transcription from an introduced Drosophila hsp70 heat shock promoter in a stress-dependent manner (Voellmy and Rungger, 1982). Thus, despite the lack of an endogenous response, the oocyte can serve as an in vivo test tube to study the induction of the stress response by microinjected proteins (Ananthan et al., 1986). This strategy was employed here. Inducer effective- ness was found to correlate with protein aggregation and lon- gevity in the cell and to depend upon nuclear rather than cy- toplasmic injection; these requirements most likely are related, as will be discussed. There is no requirement for ubiquitination of inducer proteins.

MATERIALS AND METHODS Oocytes-X. laevis oocytes were generously provided by Dr. C. Gun-

derson (Dept. of Pharmacology, UCLA). The follicle layer of individual stage V and VI oocytes was removed manually following incubation in 0 . 2 4 3 % collagenase (%e I, Sigma) in Barth's solution (87 mM NaCI, 2.4 mM NaHCO,, 0.8 mM MgSO,, 0.3 m Ca(NO,),, 0.4 m CaCl,, and 10 mM sodium HEPES, pH 7.6; Gurdon and Wickens, 1983) containing 2 mM KCl. Oocytes were stored in Barth's solution at 19 "C and injected within 2 days of defolliculation.

Protein Modification-Proteins were obtained from Sigma except for an initial sample of bovine a-crystallin, which was a gift from Dr. J. Horwitz (Jules Stein Eye Institute, UCLA). After modification, all pro- teins were exhaustively dialyzed a t 4 "C, first against water and then against Injection Buffer (10 nm sodium HEPES, pH 7.5, 68 mM NaCl). Protein solutions were sterilized with 0.2-pm cellulose acetate filters, concentrated in sterile Centricon-IO microconcentrators (Amicon Corp.), and stored frozen at -20 "C. Protein concentrations were deter- mined by the bicinchoninic acid microassay (Pierce Chemical Co.) ac- cording to manufacturer's instructions.

Protein cysteines were reduced and alkylated essentially as de- scribed by Hirs (1967). Proteins (-3 mg/ml) in a degassed buffer con- taining 6 M guanidinium chloride, 10 mM EDTA, and 1.45 M Tris-HC1, pH 8.3, were reduced with P-mercaptoethanol (twice the total cysteine concentration) for 30 min at 25 "C. Iodoacetamide or sodium iodoacetate (twice the P-mercaptoethanol concentration) was then added, and the reaction continued in the dark for -20 min. The reactions were stopped with excess p-mercaptoethanol, and the products were dialyzed as de- scribed above. [~arboxymethyl-~Hlrcm-BSA (875 cpndpg) was prepared as described above, and [carb~xymethyl-~HIrcm-a-crystallin (930 cpm/ pg) was prepared in a nondenaturing buffer (IO mM EDTA and 1.45 M

Tris-HC1, pH 8.31, with [3Hliodoacetic acid (DuPont NEN; 161.6 mCi/ mmol).

Protein lysines were reductively methylated as described (Jentoft and Dearborn, 1979). Proteins (-1 mg/ml) in 6 M urea buffered with 0.1 M sodium HEPES, pH 7.5, were mixed with a 12-fold excess (over total lysines) of formaldehyde, followed by addition of NaCNBH, to 20 m. After 4 to 5 h at room temperature, samples were dialyzed against Injection Buffer as described above. ['4C]Formaldehyde (ICN Biomedi- cals, Inc.; 56 mCi/mmol) was used to prepare [methyl-'4Clme-a-crystal- lin (400 cpdpg) . Proteins were carbamoylated in 0.1 M sodium borate, pH 8.6, containing 6 M urea (Tanaka et al., 1983). KCNO was added to 0.1 M and allowed to react with the protein (-0.5 mg/ml) for 24 h a t 37 "C. In a control treatment of a-crystallin, the KCNO was omitted from an otherwise identical reaction. Potassium ['4C]cyanate (American Radiolabeled Chemicals, Inc.; 22.1 mCi/mmol) was used to prepare [car- bamoyl-14Clcbm-a-crystallin (230 cpdpg) . BSA was iodinated as de- scribed (Wilkinson and Audhya, 1981).

Protein derivatives were characterized by amino acid analysis (Jones et al., 1981) to assess the extent of cysteine alkylation or lysine meth- ylation. Typically, 80% of the cysteines in rcm-BSA preparations were carboxymethylated, and >95% of the lysines were N-methylated in the me-rcm-BSA derivatives. For carbamoylated proteins, total free amino groups were assayed with trinitrobenzene sulfonate (Hazra et al., 1984). Because chemical denaturants interfered with this assay, complete de- naturation of the proteins could not be ensured. Assuming that all ofthe lysine residues were available for reaction with trinitrobenzene sulfon- ate, then 80% of the lysines were carbamoylated in the cbm-a-crystallin preparations.

Oocyte Injections-Injection solutions included 1.0 mg/ml plasmid p622C* in the Injection Buffer with 0.12 mg/ml [rneth~zy-~H]inulin (Du Pont NEN, 212 mCi/g). The plasmid p622C*, kindly provided by Dr. R. Voellmy (University of Miami School of Medicine), was isolated from E. coli strain MClOl according to standard protocols (Birnboim, 1983). Injection needles were pulled from soda lime glass capillary tubing, and

injections were done with a Narishige USA, Inc. IM-200 microinjector and a Singer Mark-I micromanipulator. Except where specified other- wise, injections were into the nucleus; the accuracy typically was 280% as determined from 6-galactosidase assays of individual oocytes that were thermally stressed as a positive control (see below). Injection vol- umes of -20 nl were estimated from drop diameters measured by com- parison with a hemocytometer grid. Co-injection of a radiolabeled tracer allowed for precise measurement of injection volume, as described below.

Stress Deatments and Oocyte Extract Preparation-Typically, batches of 10-20 oocytes were subjected to a given treatment. Cells were incubated in Barth's solution for 14-17 h at 19 "C following injec- tion. When cells were stressed thermally, they were incubated for 1.5 h a t 34 "C before transfer to 19 "C. Cells were inspected and transferred to 1.5-ml microcentrifuge tubes where they were washed and then ho- mogenized with 40 pUoocyte of Z Buffer (60 mM Na,HPO,, 40 mM NaH,PO,, 10 mM KCl, 1 mM MgSO,, 50 mM P-mercaptoethanol; Miller (1972)). Absolute injection volumes were determined by co-injection of [meth~xy-~Hlinulin and counting of one oocyte-equivalent of homoge- nate in 5 ml of Beckman Ready Safe liquid scintillation mixture in a Beckman LS 3133P counter. The remaining homogenate was centri- fuged at 4 "C a t 12,000 x g for 15 min, and the clear supernatant was assayed for P-galactosidase activity with o-nitrophenol-P-galactoside (Miller, 1972). o-Nitrophenol production was measured for several time points by the absorbance at 420 nm; interference from light scattering was corrected by subtracting the absorbance at 550 nm. The value for the negative control (plasmid-only injected oocytes) was subtracted, and the corrected P-galactosidase activities typically are presented relative to the positive control (plasmid injection, followed by a thermal shock).

Protein Size Characterization-For dynamic light scattering meas- urements, proteins were diluted to 2 mg/ml in Injection Buffer and transported on ice to the laboratory of Dr. E. Amis (Dept. of Chemistry, University of Southern California, Los Angeles); the samples were fil- tered (0.2 pm) immediately prior to analysis. Scattered light was de- tected a t a 90" angle and a t room temperature on an instrument equipped with a Spectra Physics 2020-3 argon-ion laser (514.5 nm, operated at 200-500 milliwatts) and a Brookhaven Instruments BI- 200SM light scattering goniometer. Photon correlation measurements were made with a 264-channel multibit autocorrelator (Brookhaven Instruments 2030) as described (Seery et al., 1989).

For gel filtration chromatography, samples of -100 pg of protein in 10-40 pl were loaded onto a 24-ml Pharmacia Superose 6 HR 10/30 column (Pharmacia Biotech, Inc.) and eluted a t room temperature with Injection Buffer at 0.4 mumin. The column was calibrated with jack bean urease (hexamer, 545 kDa; trimer, 272 kDa), thyroglobulin (669 kDa), and BSA (monomer, 66 kDa; dimer, 132 kDa), and the eluant was monitored by its absorbance at 280 nm.

Protein %mover in Vivo-Oocytes were injected with [carbamoyl- 14Clcbm-a-crystallin or [methyl-14Clme-a-crystallin or [carboxymethyl- 3Hlrcm-BSA (heated or unheated) and incubated a t 19 "C in Barth's solution. At various times, groups of six oocytes were homogenized in 180 pl of Injection Buffer containing 0.01% BSA. Two and one-half oocyte-equivalents were removed for liquid scintillation counting, and then to each remaining homogenate an equal volume of cold 30% (w/v) trichloroacetic acid was added. After 230 min at 0 "C, the samples were centrifuged at 4 "C at 12,000 x g for 15 min, and two and one-half oocyte-equivalents of supernatant were removed for counting.

RESULTS

Proteins as Stress Signals-In X . laevis oocytes injected with a plasmid containing an hsp7O promoter-lac2 fusion, expres- sion of P-galactosidase is regulated by the heat shock transcrip- tion factor and is induced by heat shock and other stresses (Voellmy and Rungger, 1982). This system provides a simple means to monitor the stress response in vivo and allows for various proteins to be evaluated as potential inducers or sup- pressors of the response following their direct injection into the cell. The idea that denatured protein, which may result from a stress condition, can serve as the signal for heat shock gene induction (Hightower, 1980; Finley et al., 1984; Goff and Gold- berg, 1985) has been tested by Ananthan et al. (1986). They reported that, of three pairs of native and chemically denatured proteins, injections of only the denatured proteins induced P-galactosidase expression in the oocyte system. Their work has been extended here to better define the features of proteins

15712 Denatured Protein Inducers of the Stress Response

rcm-BSA injected (ng per oocyte)

FIG. 1. The stress response by X. Zaeuis oocytes to nuclear in- jections of rcm-BSA is nonlinear. Oocyte injections were performed as described under “Materials and Methods.” Three different volumes of injection solution containing different concentrations of rcm-BSA were co-injected with the reporter plasmid p622C*. [metho~y-~HlInulin was included as a tracer for precise measurement of the volumes injected. P-Galactosidase activity, resulting from expression of the hsp7O pro- moter-lac2 fusion on the plasmid, is shown relative to p-galactosidase expression induced by a thermal shock. Concentrations of the protein solutions injected were 5 ng/nl (O), 7.5 nghl (01, and 10 ng/nl (A).

that may render them heat shock inducers and, in the process, to illuminate the mechanism of their recognition.

As reported by Ananthan et al. (1986), reduced and car- boxymethylated (rcm) BSA induced the stress response in Xe- nopus oocytes, whereas unmodified BSA did not. In addition to confirmation of this result, expression of p-galactosidase was found to increase gradually when up to 125 ng of rcm-BSA was co-injected with plasmid p622C* into the nucleus. Injections of even greater amounts of the denatured protein showed that the dose-response curve is distinctly nonlinear, with the stress re- sponse enhanced significantly by greater amounts of rcm-BSA (Fig. 1). Limited protein solubility and high solution viscosities, however, prevented injections of even greater amounts of pro- tein. Thus, evidence for saturation of the effect could not be obtained.

Cytoplasmic versus Nuclear Injections-To determine whether the observed effect depended on the localization of the protein, nuclear and cytoplasmic injections of an inducing pro- tein were compared. Cytoplasmic injection of the protein fol- lowed nuclear injection of the reporter plasmid p622C*. This double-injection protocol did not adversely affect oocyte sur- vival and, when followed by thermal stress as a positive control, P-galactosidase expression was induced normally. In six inde- pendent experiments, cytoplasmic injection of up to 450 ng of rcm-BSA failed to produce a detectable response, whereas in- jection of the same rcm-BSA preparations into the nucleus elic- ited high levels of P-galactosidase activity (data not shown). Ananthan et al . (1986), however, noted that they were able to obtain a stress response to cytoplasmically injected rcm-BSA. Possibly, differences in specific experimental conditions or the quality of the oocytes may be responsible for this discrepancy. From the results here, however, introduction of a stress-induc- ing protein into the nucleus is necessary to elicit a detectable response in Xenopus oocytes. All subsequent experiments employed co-injection of protein and plasmid p622C* into the oocyte nucleus.

Recognition of Stress-inducing Proteins Is Independent of Their Ubiquitination-Consistent with the idea that ubiquitin has a key role in stress response initiation, rcm-BSA, a stress- response inducer, is recognized for ubiquitination in vitro whereas native BSA, a noninducer, is not (Evans and Wilkin- son, 1985). To test whether protein ubiquitination is involved in

-1. Z m a,

m g 0.9

0 50 100 150 200 ”.

0 50 100 150 200 Protein injected (ng per oocyte)

7.0 p t 1 .- b

s

L 8 4.0

6.0 -

8 5.0 a rr -

- u

4 3.0 -

-

Protein Injected (ng per oocyte)

FIG. 2. rcm-BSA elicits a greater stress response than the N- methylated derivative, me-rcm-BSA, whereas N-methylation en- hances the response to a-crystallin. Two independent experiments are distinguished by the circles and squares. Differences between ex- periments are the result of variations among batches of oocytes and solutions of rcm-BSA (see text). Several volumes of different concentra- tions of each protein were injected as described in the legend for Fig. 1 and under “Materials and Methods.” A, the closed symbols represent rcm-BSA, and the open symbols represent me-rcm-BSA, virtually no response was observed with native BSA (x-x). B, the closed symbols represent me-a-crystallin, and the open symbols represent a-crystallin.

the selective response to rcm-BSA, lysine €-amino groups were modified completely by reductive methylation to prevent the attachment of ubiquitin. Injections of the resulting derivative, me-rcm-BSA, produced a significantly reduced p-galactosidase induction relative to rcm-BSA (Fig. 2). Although different preparations of rcm-BSA produced variable responses (see Fig. 2 and below), P-galactosidase elicited by me-rcm-BSA always was nearer the control (i.e. native BSA or no protein injected) levels. These results support the idea that recognition by the ubiquitin system is critical for stress response induction; how- ever, further experiments described below showed this not to be the case.

The effect of N-methylation was tested with a second poten- tial stress response inducer, a-crystallin, which, like rcm-BSA, can be ubiquitinated in vitro (Mayer et al., 1989). a-Crystallin is an N”-acetylated structural protein found in eye lens that undergoes increased modification in the aging eye (reviewed by de Jong et al. (1993)). As shown in Fig. 2, “native” a-crystallin from adult bovine eye elicits a moderate stress response in oocytes; possibly this is because the aged protein had under- gone covalent modifications and partial denaturation. Surpris-

Denatured Protein Inducers of the Stress Response 15713 TABLE I

Stress response induction varies with different modified forms of a-crystallin

u-Crystallin form Response (S.D.)' experimentsb No. of

Unmodified 0.03 (0.04) 8 N-Methylated 1.00 Carboxymethylated 0.56 (0.56) 8 Carboxamidomethylated 0.09 (0.07) 6 Urea-denatured 0.51 (0.45) 6 Carbamoylated 0.00 (0.00) 4

"Normalized to P-galactosidase levels elicited by injection of an equal amount of N-methylated a-crystallin; injections were of 80 to 300 ng of protein per oocyte.

Each experimental condition was tested with batches of 210 oocytes.

ingly, reductive N-methylation of lysines in a-crystallin dra- matically enhanced its ability to induce a stress response (Fig. 2). Thus, despite the results obtained with rcm-BSA and me- rcm-BSA, the stress response mechanism clearly does not de- pend upon ubiquitination of inducer proteins.

Chemical Specificity of the Stress Signal-Several modifica- tions of a-crystallin were made in order to further investigate the specificity of the stress response. Reductive methylation under denaturing conditions generated the most effective a-crystallin derivatives for P-galactosidase induction; depend- ing upon the experiment, the response was 10 to 100 times that of unmodified a-crystallin (Table I).' Denaturation in 6 M urea without additional modification produced a form of a-crystallin second only to N-methylated a-crystallin in its ability to elicit high P-galactosidase expression. This effect of urea denatur- ation on a-crystallin was reversed by carbamoylation of lysine €-amino groups. Thus, two different modifications of lysine side chains produced opposite results. Moreover, the same modifi- cation (lysine methylation) was found to have opposite effects on the abilities of two different proteins (rcm-BSA and a-crys- tallin) to induce the stress response.

In other derivatives, the single cysteine of the C+ chain of a-crystallin was modified under nondenaturing conditions. Generally, injection of reduced and carboxamidomethylated (rca) a-crystallin produced a response similar to that of a-crys- tallin, whereas the response to rcm-a-crystallin was highly variable. This variability could be due to the sensitivity of the denatured proteins to freeze and thaw treatments and will be discussed later.

Many Denatured Proteins Fail to Induce the Stress Response in Oocytes-Because only some of the forms of BSA and a-crys- tallin induced the stress response, additional proteins were tested as potential stress response inducers (Table 11). Contrary to the report of Ananthan et al. (1986), in four of five experi- ments, no significant @-galactosidase activity was detectable after injection of rcm-P-lactoglobulin, and no response was ob- served when the protein was further modified by either N- methylation or carbamoylation. Iodinated BSA, a-casein, and oxidized RNase A (all ubiquitination substrates in vitro; Wilkinson and Audhya (1981) and Ferber and Ciechanover (198611, modified bovine y-globulins, and N-methylated oxi- dized RNase A all failed to induce the stress response. Ly- sozyme, Cys6/Cys12'-reduced and carboxymethylated lysozyme (Hill et al., 1993), apocytochrome c, actin, and several different preparations of globin were insoluble in our injection system and could not be tested. Nevertheless, it is clear that neither denaturation of a protein nor its ability to be ubiquitinated is sufficient for stress response induction.

Variability of rem-BSA Preparations as Stress Response

This variation is due partly to differences in the protein stocks, but also to variations among batches of oocytes.

TABLE I1 Not all denatured proteins induce a stress response when

injected into oocytes

Protein injected' Stress responseb No. of experiments'

BSA - 250 rcm-BSA ++d

me-rcm-BSA 250

-I+ I-BSA

240 - 1 P-Lactoglobulin - 5 rcm-P-Lactoglobulin - 5 me-rcm-0-Lactoglobulin - 4 cbm-rcm-p-Lactoglobulin - 4 oxRNase A - 1 me-oxRNase A - 1 a-Casein - 7 a-Crystallin' + 220 me-a-Crystallin' +++ 220

Injections contained 100-400 ng of protein per oocyte. Determined as 0-galactosidase expression; a thermal shock typi-

E Each experiment usually employed 2 3 protein concentrations, with cally would score ++.

batches of 10-20 oocytes for each. Vaned, depending upon protein preparation (see text). This protein was found to induce the stress response by Ananthan

et al. (1986). Globin, also reported to induce a response, was insoluble in the injection solution and could not be tested.

'Also see Table I for quantitation on these and related derivatives.

TABLE 111 Heating samples of rcm-BSA reduces its ability to induce the

stress response

Experiment Heated" (relative to a thermal shock) P-Galactosidase activity

1 - 0.08

2 - 0.06

3 0.01 - 0.14

+ 0

+ 0

+

"In three independent experiments, samples of rcm-BSA were heated for 2 min at -80 "C and cooled to room temperature before preparation of the injection mixture; 76 to 106 ng of protein were in- jected per oocyte.

Inducers-Some samples of rcm-BSA were observed to have atypically weak inducer activity. Amino acid analysis and SDS- gel electrophoresis failed to distinguish rcm-BSA preparations that were good and poor inducers. The variability instead cor- related with the viscosity of the rcm-BSA solutions and their propensity to gel, a property that set rcm-BSA apart from the other proteins studied. Concentrations of greater than -30 mg/ml during preparation or repeated freeze-thaw cycles occa- sionally caused gelation of the entire rcm-BSA stock solution; resolubilization could be accomplished by a brief heat treat- ment. Preheating the protein stock lowered the solution viscos- ity as evidenced by increased flow through the injection mi- cropipet relative to that of the protein prior to heating, but rcm-BSA samples treated this way also lost their ability to stress the oocytes. A single heat treatment to a gelled stock of rcm-BSA was sufficient to suppress stress response induction. Four subsequent freeze-thaw cycles and a step to concentrate the stock 2-fold partially restored the protein's inducing capa- bility, but only to 3% of its original level. In three separate experiments, an aliquot of this protein solution was heated again (80 "C, 2 to 3 min), suppressing its already reduced ac- tivity (Table 111). I t would appear that heating may have per- manently affected the protein, except that the stress-inducing activity of the same protein used in Table 111, with two addi- tional lower-temperature (60 "C, 3 min) heat treatments, was fully restored upon concentration to its original level (data not shown).

15714 Denatured Protein Inducers of the Stress Response

I I I I

80 t

Diameter (nm)

FIG. 3. Reduced and carboxymethylated (rcm) BSAforms large aggregates in solution that disassemble upon heating. Proteins were prepared for dynamic light scattering measurement of particle size distribution as described under "Materials and Methods." A , BSA, B, rem-BSA, C, rcm-BSA that was heated for approximately 2 min at 80 "C before analysis.

An explanation that accounts for these observations is that the state of aggregation of rcm-BSA is related to its ability to induce the stress response. The difficulty of injections due to the solution viscosity correlated with the stress response throughout the rcm-BSA experiments. Multiple cycles of freez- ing and thawing the stock solution of rcm-BSA also effected the physical state of the protein. Freezing the rcm-BSA stock solu- tion temporarily concentrates the protein and, because the pro- tein tends to aggregate, caused increasingly larger aggregates to form that did not dissociate readily at room temperature. Heating resolubilized the protein and reduced its ability to induce the stress response. If the inducer activity of rcm-BSA correlates with increased aggregation, then freezing and thaw- ing would increase its effectiveness (until it would be too highly aggregated to be injected), and heating would decrease the activity depending upon the extent to which the protein is disaggregated. In this manner, the heat treatments could have had variable effects on the quality of the protein as a stress response inducer. Interestingly, the only other proteins found that consistently produced a stress response were forms of cy-crystallin, which is known to form large aggregates in solu- tion (de Jong et al., 1993). The physical properties of inducer proteins were investigated to study this correlation further.

Particle Size Analysis of rcm-BSA by Dynamic Light Scattering-Before analysis, all proteins were diluted (5-fold to 2 mg/ml) and passed through 0.2-pm cellulose acetate filters to remove dust particles. The rcm-BSA solution was the most difficult to filter, even after dilution, indicating that aggrega- tion of rcm-BSA is not rapidly reversed upon dilution. Heating the rcm-BSA (2-3 min, -80 "C) just before dilution, however, rendered it easy to filter. The results of dynamic light scatter- ing (DLS) measurements are shown in Fig. 3. Note that be-

TABLE IV Distribution of BSA and modified BSA aggregates estimated by

gel filtration on Superose 6

Number of Percentage of protein in each size range Size range' monomers

per particleb BSA rcm-BSA rcm-BSA' me-rcm-BSA

kDa -70 1 79 3 3 1 -130 2 13 3 4 3 -200 3 7 10 10 6 -270 4 1 3 14 23

290-380 4-6 3 6 16 380-500 6-8 4 8 8 500-850 8-13 7 15 19 850-1,400 13-21 13 13 13

1,400-2,400 21-37 16 15 10 22,400 237 38 13 2

a Estimated from a semilog plot of molecular mass Versus elution volume of protein standards (see "Materials and Methods").

* Calculated assuming that the particles are spherical. The rcm-BSA was heated for 2 min at -80 "C before chromatogra-

phy.

cause the intensity of scattered light by a particle is propor- tional to the particle radius cubed (Nicoli et al., 19911, larger particles give a significantly higher signal than equal numbers of smaller particles.

With BSA, the peak of 7 nm diameter particles reflects a weighted average of BSAmonomers and dimers which have not been successfully resolved in the DLS analysis (Fig. 3A). Na- tive polyacrylamide gel electrophoresis using 6% acrylamide gels indicated that 20-30% of the unmodified BSA was dimeric (data not shown). This suggests that the DLS peak is skewed to 7 nm by the disproportionately greater scattering intensity of the dimers. In the case of rcm-BSA, free monomers are appar- ent as particles of 3-4 nm in diameter (Fig. 3B). Most signifi- cantly, the rcm-BSA preparation contained particles with di- ameters as great as 110 nm. Although these large aggregates may represent a small number of particles in solution, they contain a large fraction of the total rcm-BSA protein. Preheat- ing the rcm-BSA stock solution dramatically altered the par- ticle distribution (Fig. 3C) so that, although approximately half of the rcm-BSA protein remained in large aggregates, a popu- lation of smaller species had arisen with an average diameter of 14 nm (60-70 monomers per particle). Thus, the stress re- sponse induction by rcm-BSA correlates with the extent of its aggregation.

Size Exclusion Chromatography of Injected Proteins-The DLS results with rcm-BSA were confirmed by gel filtration on a Superose 6 FPLC column (Table IV). This method comple- mented the DLS measurements in that it is more sensitive for the detection of smaller particles. Again, the aggregate size of rcm-BSA correlated with its ability to induce the stress re- sponse, and the population of protein aggregates shifted to smaller species upon heating. Prior to heating, nearly 40% of the protein migrated in the void volume of the column (25 x lo6 Da), and the next smaller species, representing 16% of the protein, corresponded to a molecular mass of greater than 1.4 x lo6 Da; only 5 4 % of the rcm-BSA was found as monomers and dimers (Table IV). Heating converted half of the largest par- ticles to species containing fewer than an estimated 13 rcm- BSA monomers, although monomers and dimers still repre- sented a minor portion (6%) of the total. Thus, the most substantial change upon heating the rcm-BSA was a shift from large to moderate-sized aggregates, and this shift extended to the largest aggregates, as evidenced by DLS.

me-rcm-BSA, which produces virtually no stress response in injected oocytes, has a population further skewed toward smaller aggregates (Table IV). Less than 2% of me-rcm-BSA was excluded from the Superose 6 column, 32% of the protein

Denatured Protein Inducers of the Stress Response 15715 TABLE V

Size distribution of a-crystallin and modified a-crystallin aggregates by Superose 6 gel filtration

Molecular size ,&:;Ef Percent of total protein range“ monomersb a-Crys me-a-Crys den-a-Crys‘ cbm-a-Crys

kDa 11-37 1-2 6 1 1 1 37-120 2-6 1 1 2 5

1 2 M 1 0 6 2 0 5 5 5 35 410-1400 20-70 74 56 49 53

14004500 70-225 12 28 37 7 4500-15,000 2225 3 8 6

Estimated from a semilog plot of molecular mass uersus elution volume of protein standards (see “Materials and Methods”).

* Calculated assuming that the particles are spherical. den-a-Crys is a-crystallin that was denatured by incubation in 6 M

urea.

formed particles of between 0.5 x lo6 and 1.4 x lo6 Da, and one-half of the me-rcm-BSA formed hexameric or smaller ag- gregates. Iodinated BSA, which fails to evoke a response in oocytes (Table II), had a gel filtration profile identical with that of the native protein and showed no large aggregates (data not shown). It appears, then, that for BSA and its derivatives, the extent of aggregation correlates with the ability of the protein to induce the stress response, where formation of the largest particles (21.4 x lo6 Da) seems to be critical.

Sizes of a-Crystallin Derivatives-a-Crystallin forms large aggregates in solution (de Jong et al., 1993). Sizes reported for these aggregates vary according to protein concentration, buffer components, temperature, and other conditions, and it is possible that the results from size exclusion chromatography here may have been influenced by interaction with or dilution on the Superose 6 column. Freeze and thaw treatment of the protein stock affected the size distributions of these popula- tions as well (data not shown) and may account for some of the variability in the magnitude of the stress response; however, these changes were not easily controlled for systematic study by microinjection. Gel filtration of a-crystallin and its deriva- tives on a Superose 6 column revealed that all formed very large aggregates in Injection Buffer. Nonetheless, nearly 30% of me-a-crystallin and 40% of urea-denatured a-crystallin formed aggregates of >1.4 x lo6 Da, whereas only 14% of unmodified a-crystallin and 7% of cbm-a-crystallin contained aggregates this large (Table V). This distribution of larger aggregates cor- relates with the ability of the protein to induce a stress re- sponse when injected into oocytes (Table I). rca-a-Crystallin is an exception, however, since it forms large aggregates but pro- duced only 2-15% of the response elicited by me-a-crystallin (Table I). Gel filtration indicated that rca-a-crystallin and urea- denatured a-crystallin have similar sizes (data not shown), although urea-denatured a-crystallin usually induced a greater stress response when injected into oocytes. This exception to the correlation between size and oocyte stress induction could reflect the lack of a denaturation step during the preparation of rca-a-crystallin or the relatively minor nature of the modifica- tion (a single cysteine on only one of the two a-crystallin polypeptide chains is available for alkylation). Perhaps rca-a- crystallin retains a more native-like structure that, in spite of its aggregation, less effectively elicits a stress response. Al- though aggregation is related to a protein’s ability to induce a stress response, induction may depend as well on other prop- erties of the protein.

In Vivo Degradation of Injected Proteins-Degradation of in- jected proteins was monitored in order to determine whether stress response induction by a protein relates to its longevity in the cell. Although injections of “C-labeled me-a-crystallin and cbm-a-crystallin gave somewhat variable stress responses with

h I I

Time After Injection (h) FIG. 4. [carb~rnqyZ-’~C]cbm-a-Crystallin is degraded in vivo at

a higher rate than [methyZ-’4C]me-a-cryystallin. Oocytes were in- jected and extracts were prepared as described under “Materials and Methods.” Degraded protein was determined as the fraction of radioac- tivity soluble in 15% trichloroacetic acid. The results shown are repre- sentative of five independent experiments. Here, injections of 310 ng of [rnethyl-’4Clme-a-crystallin (0) per oocyte produced p-galactosidase ac- tivity of 0.32 (relative to a thermal shock); no p-galactosidase was elic- ited by the injections of 370 ng of [carbarn~yl-~~Clcbm-a-crystallin per oocyte (0). Note that both proteins are degraded similarly during the first few hours after injection, but that this rapid phase of turnover persists longer with the cbm-a-crystallin derivative.

different batches of oocytes, a clear difference in their degra- dation rates was observed. By 15 h after injection, the cbm-a- crystallin that remained was only about one-fourth that of the me-a-crystallin (Fig. 4). Differences in the intrinsic rates of proteolysis of the two chemically distinct derivatives could be responsible. Alternatively, if the proteolysis is cytoplasmic and similar for both proteins, then the data suggest that the more highly aggregated (and stress-inducing) protein may be re- tained longer in the oocyte nucleus. Each protein forms a wide range of aggregates (Table V). If particles of similar size exit the nucleus at the same rate, differences in the degradation curves manifested at later times then could reflect the slower passage (or solubilization) of the largest aggregates. Degradation stud- ies that compare cytoplasmic and nuclear injections of proteins having better defined sizes are needed to explore this issue further.

The longer that an aggregated protein is in the nucleus, the greater are its opportunities to interact with, and possibly dam- age, other nuclear proteins. Alternatively, the rate of solubili- zation or transport of the aggregated protein through the nuclear pore may account for both its slow turnover and its ability to induce the stress response. That the critical difference is the rate at which the proteins exit the nucleus and are degraded by cytoplasmic proteases would account for the ap- parent delay before significant differences in degradation were observed. The results in Fig. 4 support the idea that the more highly aggregated protein is retained better within the nucleus, and that the longevity of the denatured protein in the nucleus may contribute to an increased stress response.

Interpretation of the different degradation rates of the two a-crystallin derivatives is complicated by their different chemi- cal modifications. This complication can be eliminated by capi- talizing on the differential stress response induced by rcm-BSA and heated rcm-BSA. Heated rcm-BSA, which gives a lower response than its unheated counterpart (Table 1111, is degraded more quickly (Fig. 5). Again, this is consistent with the idea that the less effective inducer (in this case the heated, more soluble form of rcm-BSA) is more quickly eliminated from the

15716 Denatured Protein Inducers of the Stress Response

Y

2 0 ~ ~ ' " " " " " 0 2 4 6 0 10 12

l ime After Injection (h) FIG. 5. Heated [~arb~methyZ-~Hlrcrn-BSA is degraded more

quickly than unheated [~arb~methyZ-~HIrcm-BSk Experimen- tal procedures were as described in the legend to Fig. 4. The unheated sample of ~carb~xymethyl-~H]rcm-BSA (0) produced 110% of the P-pa-

the sample heated for -2.5 min at 80 "C (0) elicited only 10% of the lactosidase activity of the positive, thermally stressed control, whereas

P-galactosidase activity as the positive control. Injections contained approximately 70 ng of protein per oocyte.

nucleus and made available for proteolysis. Although the dif- ference in degradation rates is modest (50% of the heated rcm- BSA was degraded after -4 h versus 8.5 h for the unheated protein), the much greater stress response elicited by unheated rcm-BSA is quite reasonable given the dramatic nonlinearity of the response (see Fig. 1)

DISCUSSION

The direct introduction into X. laeuis oocytes of a chemically denatured protein, cysteine-S-carboxymethylated BSA (rcm- BSA), elicited a stress response, whereas native BSA did not (Ananthan et al . , 1986). These results were reproduced here, and, additionally, greater amounts of denatured protein were found to produce a disproportionately greater response. This nonlinear behavior may reflect either the initial portion of a simple titration curve or cooperative binding. Stress response induction by a-crystallin was similar, and also may depend upon protein denaturation since this protein accumulates sub- stantial covalent damage in the aging eye lens (de Jong et al., 1993).

In eukaryotic cells, damaged proteins can be substrates for ubiquitination. Potentially, such ubiquitin-protein conjugates could trigger a stress response by the cell. Both a-crystallin and rcm-BSA, which induce a stress response when injected into oocytes, are ubiquitination substrates in vitro (Mayer et al . , 1989; Evans and Wilkinson, 1985). However, that several ubiq- uitination substrates were ineffective as inducers (e.g. oxRNase A, I-BSA, rcm-p-lactoglobulin, a-casein) suggested that the ability to be ubiquitinated is not sufficient, although the pos- sibility remained that it was a necessary property of a protein inducer. Modification of the lysine €-amino groups of rcm-BSA and a-crystallin therefore was done to prevent their ubiquiti- nation. Whereas methylation of the lysines in rcm-BSA signifi- cantly reduced its ability to elicit the stress response, meth- ylation of a-crystallin actually enhanced the oocyte stress response over that of unmethylated a-crystallin. Thus, ubiquit- inability is not an essential property of protein stress response inducers.

Stress response induction by denatured proteins could in- volve recognition of specifically modified amino acids or expo- sure of sequences normally buried within hydrophobic regions.

Several protein derivatives were tested to determine whether particular chemical modifications would consistently generate stress response inducers. Whereas N-methylation of a-crystal- lin lysines greatly enhanced the stress response, carbamoyla- tion of lysines eliminated it. Moreover, N-methylation had op- posite effects with rcm-BSA and a-crystallin. Thus, neither specific amino acids nor the nature of a chemical modification is particularly critical for stress response induction.

Many denatured proteins failed to stress injected oocytes under conditions in which a-crystallin and rcm-BSA were ef- fective. Interestingly, whereas urea denaturation of a-crystal- lin increased its inducer activity and this increase was main- tained upon methylation of lysines (me-a-crystallin), carbamoylation of lysines in urea-treated a-crystallin com- pletely eliminated the protein's ability to induce a response. It is unlikely that carbamoylation renatured the protein, and, in fact, the stress response to cbm-a-crystallin was lower than to the unmodified protein (Table I). In principal, the neutraliza- tion of positive charges by carbamoylation could have altered the a-crystallin structure to conceal a critical site on the pro- tein. However, exposure of internal amino acids does not ap- pear to be sufficient for stress induction. For example, oxRNase A is essentially a random coil in solution (Harrington and Sela, 19591, yet it failed to produce any response. Several other pro- tein derivatives, all denatured to various extents, also had no effect (Table 11); thus, denaturation alone of the injected protein is insufficient to induce a stress response.

Unexpectedly, and in contrast with a published report (An- anthan et al . , 19861, proteins that induced a stress response when injected into the nucleus produced no detectable response when injected cytoplasmically. Although the cytoplasmic injec- tions contained more protein than was injected into the nucleus, dilution into the cytoplasm, which has an -20-fold greater volume than the nucleus, could account for a lowered response. However, as little as 30 ng of rcm-BSA injected into the nucleus elicited 2.0% of the P-galactosidase found in ther- mally stressed control cells; this activity was 5 times greater than in the unstressed control cells and readily measured. Thus, dilution of the inducer into the cytoplasm should not have precluded a detectable response. A possible explanation for the failure of cytoplasmically injected rcm-BSA to produce a response comparable to the nuclear injections could be that the large amount of yolk protein present in X. laevis stage V and VI oocytes masked the denatured protein from interactions with critical cytoplasmic factors. Alternatively, the recognition fac- tor(s) for stress activation could be located in the nucleus and must interact directly with the inducing protein. The major transcription factor responsible for heat shock gene initiation, however, is found in both the cytoplasm and nucleus of un- stressed cells (Sarge et al., 1993). Thermal shock and most other stress treatments effect proteins either already in the nucleus or able to translocate to the nucleus. Experiments that introduce denatured protein selectively into either the nucleus or the cytoplasm are necessary in order to better assess the role of stress response inducer localization. Whether this apparent requirement for nuclear localization is general or is particular to the Xenopus oocyte is unknown.

Several modifications of proteins produced a range of stress responses that were generally reproducible for a given deriva- tive. However, induction by rcm-BSA was unusually variable. The difficulty of injecting rcm-BSA solutions, due to a propen- sity of the protein to aggregate, correlated with the ability to stress the oocytes. Another inducer, a-crystallin, forms large aggregates in solution (de Jong et al . , 19931, and actin and globin, which were reported (Ananthan et al . , 1986) to induce the stress response in oocytes, formed insoluble aggregates to such a degree that, in this study, their microinjection was not

Denatured Protein Inducers of the Stress Response 15717

possible. Proteins that did not aggregate did not induce a stress response.

Modulation of stress response induction observed with heated rcm-BSA solutions allowed for a comparison of the physical state of the protein with its stress-inducing properties. Heating rcm-BSA not only reduced its ability to stress the oocytes, but also shifted the fraction of protein found in large aggregates to smaller particles (see Fig. 3 and Table IV). Simi- larly, with a-crystallin derivatives and other modified forms of BSA, the proportion of protein found in the largest aggregates correlated with the ability of the protein to induce a stress response. Approximately 50% of rcm-BSA and 40% of me-cy- crystallin and urea-denatured a-crystallin were in these large aggregates. This correlation with inducer effectiveness ex- tended to particles of 24.5 x lo6 Da. Comparison between me- a-crystallin, urea-denatured cu-crystallin, and rcm-BSA reveals that their relative activities are similar on a per mass of protein injected, rather than a per mole, basis. me-rcm-BSA and cbm- a-crystallin, which induced little or no stress response, also contained large aggregates (100-1400 kDa, representing 50- 80% of the protein), but only minor amounts were in particles of >14OO kDa. Thus, aggregation per se is insuffkient for stress response induction, and the extent of aggregation of the protein may be critical. This could reflect specific interactions that are also recognized by the stress response recognition machinery. Aggregation is thought to result from specific interactions be- tween partially folded proteins (Mitraki and King, 19891, and the degree of aggregation may reflect the quality of these in- teractions. Proteins that aggregate extensively may bind better to a factor critical for stress response induction.

A minimum aggregate size for protein inducers suggests that retention by a physical barrier, such as a nuclear pore, might be involved in efficient stress response induction. The importance of inducer clearance from the nucleus is further implicated by the apparent requirement for nuclear injections. Degradation rates of cbm-a-crystallin uersus me-a-crystallin and heated uer- sus unheated rcm-BSA indicated that the stress response cor- related with inducer lifetime in the cell. It is unclear whether the stress response to an inducer protein interferes with its degradation or whether the increased longevity of the inducer protein is integral to its stimulation of the response. Alterna- tively, the reduced degradation could reflect a rate-limiting step for solubilization of the inducer proteins or transport out of the nucleus. Particles up to 200-250 A in diameter can pass through the nuclear pore (Feldherr et al., 1984; Mehlin et al., 1992). This suggests that active transport of the smaller aggre- gates of modified BSA and cy-crystallin, inducers and non-in- ducers alike, is not directly related to stress response induction. Larger aggregates such as the 500-A particles of rcm-BSA (see Fig. 3B) are likely to require processing prior to their transport through the nuclear pore complex. Granules of aggregated pro- tein have been noted in stressed cells (e.g. Pelham (1984), Nover et al. (1989), Dubois et al, (1991), Beckmann et al. (1992)). Proteins in these granules gradually regain activity hours after recovery from a thermal stress (Dubois et al., 1991; Pinto et al., 1991). hsp70, which is responsible for solubilization of partially denatured proteins (Pelham, 1984; Lewis and Pel- ham, 19851, also participates in protein transport into and out of the nucleus (Shi and Thomas, 1992). hsp7O (or hsc7O) may be

involved in the recognition of stress response inducer proteins in either or both of these capacities. Moreover, the nonlinear increase in the stress response obtained from increasing amounts of injected inducer proteins is consistent with the titration of a factor that regulates hsp gene expression. Evi- dence that hsp7Ohsc70 proteins serve this key recognition function is presented in the accompanying paper (Mifflin and Cohen, 1994).

the oocytes, Dr. Eric Amis for assistance with the dynamic light scat- Acknowledgments-We thank Dr. Cameron Gunderson for supplying

tering measurements, Dr. Richard Voellmy for the original sample of plasmid p622C*, Dr. Joseph Honvitz for a sample of a-crystallin, and Dr. Peter Rubenstein for helpful comments on the manuscript.

REFERENCES

Ananthan, J., Goldberg, A. L., and Voellmy, R. (1986) Science 232, 522-525 Beckmann, R. P., Lovett, M., and Welch, W. J. (1992) J. Cell Biol. 117, 1137-1150

Bond, U., and Schlesinger, M. J. (1985) Mol. Cell. B i d . 5, 949-956 Birnboim, H. C. (1983) Methods Enzymol. 100,243-255

Bond, U.,Agell, N., Haas, A. L., Redman, K, and Schlesinger, M. J. (1988) J. B i d .

Ciechanover, A., Finley, D., and Varshavsky, A. (1984) Cell 37, 57-66 Craig, E. A., and Gross, C. A. (1991) '?Fends Biochem. Sci. 16, 135-140 de Jong, W. W., Leunissen, J. A. M., and Voorter, C. E. M. (1993) Mol. B i d . Euol. 10,

Dubois, M. F., Hovanessian, A. G., and Bensaude, 0. (1991) J. Biol. Chem. 266,

Evans, Jr., A. C., and Wilkinson, K. D. (1985) Biochemistry 24,2915-2923 Feldherr, C. M., Kallenbach, E., and Schultz, N. (1984) J. Cell Biol. 99,2216-2222 Ferber, S., and Ciechanover, A. (1986) J. Biol. Chem. 261, 3128-3134 Finley, D., Ciechanover, A., and Varshavsky, A. (1984) Cell 37 ,4345 Finley, D., Ozkaynak, E., and Varshavsky, A. (1987) Cell 48, 1035-1046 Goff, S. A,, and Goldberg, A. L. (1985) Cell 41, 587-595

Harrington, W. F., and Sela, M. (1959) Biochim. Biophys. Acta 31,427-434 Gurdon, J. B., and Wickens, M. P. (1983) Methods Enzymol. 101, 370-386

Hazra, A. K., Chock, S. P., and Albers, R. W. (1984)AnaL Biochem. 1 3 7 , 4 3 7 4 3 Hightower, L. E. (1980) J. Cell. Physiol. 102, 407-427 Hightower, L. E. (1991) Cell 66, 191-197 Hill, C. P., Johnston, N. L., and Cohen, R. E. (1993) Proc. Natl. Acad. Sci. U. S. A.

Hiromi, Y., Okamoto, H., Gehring, W. J., and Hotta, Y. (1986) Cell 44,293-301

Jentoft, N., and Dearborn, D. G. (1979) J. Bid. Chem. 254,43594365 Hirs, C. H. W. (1967) Methods Enzymol. 11, 199-203

Jones, B. N., Paabo, S., and Stein, S. (1981) J. Liq. Chromatogx 4, 565-586 King, M. L., and Davis, R. (1987) Deu. B i d . 119, 532-539 Lewis, M. J., and Pelham, H. R. B. (1985) EMBO J. 4, 3137-3143 Lindquist, S.. and Craig, E. A. (1988) Annu. Reu. Genet. 22, 631477 Lis, J. T., and Lee, H. (1988) in The Ubiquitin System (Schlesinger, M. J., and

Hershko, A,, eds) pp. 56-61, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Mayer, A., Siege], N. R., Schwartz, A. L., and Ciechanover, A. (1989) Science 244, 1480-1483

Mehlin, H., Daneholt, B., and Skoglund, U. (1992) Cell 69, 605-613 Mifflin, L. C., and Cohen, R. E. (1994) J. Biol. Chem. 269,15718-15723 Miller, J . H. (1972) Experiments in Molecular Genetics, pp. 352-355, Cold Spring

Mitraki, A,, and King, J. (1989) BiolTechnology 7, 690-697 Nicoli, D. F., McKenzie, D. C., and Wu, J . 3 . (1991)Am. Lab. (Shelton) 23, 32-40 Nover, L. (1984) Heat Shock Response of Eukaryotic Cells, pp. 8-12, Springer-

Nover, L., Scharf, K.-D., and Neumann, D. (1989) Mol. Cell. Bid . 9, 1298-1308 Okamoto, H., Hiromi, Y., Ishikawa, E., Yamada, T., Isoda, K, Maekawa, H., and

Parsell, D. A,, and Sauer, R. T. (1989) Genes & Deu. 3, 1226-1232 Pelham, H. R. B. (1984) EMBO J. 3,3095-3100 Pinto, M., Morange, M., and Bensaude, 0. (1991) J. B i d . Chem. 266,13941-13946 Sarge, K. D., Murphy, S. P., and Morimoto, R. I. (1993) Mol. Cell. Bid. 13, 1392-

Schlesinger, M. J. (1990) J. Biol. Chem. 266, 12111-12114 Seery, T. A. P., Shorter, J. A,, and Amis, E. J. (1989) Polymer 30, 1197-1203 Seufert, W., and Jentsch, S. (1990) EMBO J. 9, 543-550 Shi, Y., and Thomas, J. 0. (1992) Mol. Cell. Biol. 12, 2186-2192 Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585 Voellmy, R., and Rungger, D. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1776-1780 Wilkinson, K. D., and Audhya, T. K. (1981) J. Bid . Chem. 256, 9235-9241

Chem. 263,2384-2388

103-126

9707-9711

90,4136-4140

Harbor Laboratory Press, Cold Spring Harbor, NY

Verlag, Berlin

Hotta, Y. (1986) EMBO J. 5, 589-596

1407