JOURNAL OF VIROLOGY, May 2002, p. 4233–4240 Vol. 76, No. 90022-538X/02/$04.00�0 DOI: 10.1128/JVI.76.9.4233–4240.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Ty1 Defect in Proteolysis at High TemperatureJoseph F. Lawler, Jr.,1 Daniel P. Haeusser,2† Angie Dull,2‡ Jef D. Boeke,1 and Jill B. Keeney2*

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,1

and Department of Biology, Juniata College, Huntingdon, Pennsylvania 166522

Received 11 July 2001/Accepted 15 January 2002

Retrotransposition of the Ty1 element of Saccharomyces cerevisiae is temperature sensitive. Transpositionactivity of Ty1 is abolished at temperatures above 34°C. In this report, we show that the major block totransposition at high temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and theconcomitant reduction of reverse transcriptase (RT) activity. Expression of a Ty1 protease construct inEscherichia coli shows that protease enzymatic activity is inherently temperature sensitive. In yeast, Gagprocessing is only partially inhibited at high temperature, while cleavage of the Gag-Pol polyprotein iscompletely inhibited. Sites of proteolytic processing are differentially susceptible to cleavage during growth athigh temperature. Overall levels of the Gag-Pol polyprotein are reduced at high temperature, although theefficiency of the requisite �1 frameshifting event appears to be increased. RT activity is inherently relativelytemperature resistant, yet no cDNA is made at high temperature and the amount of RT activity is greatlyreduced in virus-like particles formed at high temperature. Taken together, these results suggest that alter-ations in Ty1 proteins that occur at high temperature affect both protease activity and RT activity, such thatTy1 transposition is abolished.

The Ty1 element of the yeast Saccharomyces cerevisiae is a6-kb retrotransposon with long terminal repeats (LTRs) ateach end (4). Transcription of the element is directed by the 5�LTR and produces an RNA molecule from which the element-encoded proteins Gag and Gag-Pol are translated. Host pro-tein Spt3p has been shown to be required for Ty1 elementtranscription (30). These proteins are subsequently assembledinto structures termed virus-like particles (VLPs) and pro-cessed by the element-encoded protease (PR). VLPs are es-sential replication intermediates in which reverse transcriptionoccurs (12, 14).

The 49-kDa Gag protein is the most abundant translationalproduct and is the major structural determinant of VLPs (25).A �1 frameshift signal 15 nucleotides (nt) 5� to the stop codonfor Gag results in the production a smaller amount of a 199-kDa Gag-Pol fusion protein (2, 7). This fusion protein containsthe Gag structural protein, as well as the enzymes PR, inte-grase (IN), and reverse transcriptase (RT)/RNase H (1, 12, 24,32).

PR processes both the Gag and the Gag-Pol translationproducts (Fig. 1). Cleavage of the C-terminal 40 amino acids ofGag yields a processed Gag-p45 (CA) protein. The Gag-Polpolyprotein undergoes a semiordered cleavage by PR, releas-ing each of the protein components (Fig. 1). The Gag-PRcleavage site portion is hydrolyzed first, at the same location atwhich the smaller Gag-p49 protein is processed. This step isessential, and blocking this cleavage inhibits all further pro-cessing (21, 22). The remaining p160-Pol polyprotein is cleavedto release PR, IN (90 kDa), and RT/RNase H (60 kDa). Dur-

ing the assembly process, the Ty1 RNA is packaged within theVLPs and subsequently reverse transcribed into a full-lengthcDNA. In the final step of transposition, the cDNA is inte-grated into a new site in the host genome, and the cycle canbegin anew with transcription of the newly transposed element.Although they employ a virus-like replication process, Ty1VLPs are noninfectious and do not leave the host cell. Previousstudies indicated that proteolytic processing was regulated incells and that overproducing Ty1 mRNA overcomes a defect inprocessing by Ty1 (9).

Growth at high temperature has a multitude of cellular con-sequences, and it has been known for some time that Ty1transposition is inherently temperature sensitive (5, 26). Trans-position frequency is maximal at 22°C and is still readily de-tectable at 30°C, the standard growth temperature for S. cer-evisiae. At higher temperatures, the level of transpositiondrops rapidly, and transposition is abolished above 34°C. Inthis report, we show that the major block to transposition athigh temperature is the inhibition of processing of the Gag-Pol-p199 polyprotein and a concomitant reduction of RT ac-tivity. Autoprocessing of a Gag-PR fusion protein expressed inEscherichia coli is also inhibited at high temperature. Corre-spondingly, in yeast, no processing of the Gag-Pol-p199 isdetected at high temperature. Cleavage of the Gag-p49 proteinis variable and is slightly reduced at high temperature in somestrains, indicating variability in the temperature sensitivity ofthe various processing sites. No cleavage of the Gag-p49 pro-tein is seen in a PR� mutant grown at high temperature.Previous studies of PR cleavage site mutants have shown thatthe processing of the Gag/PR site is a prerequisite to furtherprocessing (21). At high temperature, the Gag-Pol polyproteinyield is reduced and the processing of this polyprotein pheno-copies a PR� active-site mutant. Ty1 cDNA synthesis is unde-tectable at high temperature, which is further evidence for avirtually complete Gag-Pol processing defect. Although exog-enous RT activity is not innately temperature sensitive, the RT

* Corresponding author. Mailing address: Department of Biology,Juniata College, 1700 Moore St., Huntingdon, PA 16652. Phone: (814)641-3577. Fax: (814) 641-3685. E-mail: keeney@juniata.edu.

† Present address: Molecular Cell Biology Program, WashingtonUniversity School of Medicine, St. Louis, MO 63110.

‡ Present address: Center for Molecular Toxicology and Carcino-genesis, Pennsylvania State University, University Park, PA 16801.

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activity in VLPs formed at high temperature is greatly reduced.Thus, we propose that alterations in Ty1 proteins that occur athigh temperature affect both PR activity and RT activity, suchthat Ty1 transposition is completely blocked.

MATERIALS AND METHODS

Yeast strains. The yeast strains and Ty1 plasmids used for transposition assaysand biochemical analysis of VLPs are given in Table 1.

Transposition assays. For quantitation, strains were initially grown as �12- by12-mm patches on SC-Ura medium to maintain plasmid selection. Cells werethen replica plated to galactose medium and incubated at the appropriate tem-perature for 44 to 48 h to induce transposition. Following galactose inductionpatches were printed to SC-His medium. The cells remaining on the galactoseplates were transferred to 10 ml of sterile water (dilution 1). Fifty microliters ofthis dilution was transferred to 5 ml of sterile water (dilution 2). One hundredmicroliters of dilution 1 was plated to SC-His medium, except at temperaturesabove 32°C, in which case cells in dilution 1 were pelleted, resuspended in �200�l of water, and plated to SC-His. Fifty microliters of dilution 2 was plated toyeast extract-peptone-dextrose (YPD). Following incubation at 30°C (YPD, 2days; SC-His, 4 days) the resulting colonies were counted. Transposition fre-quency was calculated by dividing the number of colonies on SC-His by the totalnumber of cells plated on SC-His, as determined by the colony number on YPD,factoring in the dilution and original volume of dilution 1 plated. If no coloniesappeared on SC-His, transposition frequency was taken to be less than thefrequency calculated from a single colony.

Cell homogenates. The cell growth procedure was based on a protocol previ-ously described (22). Specifically, cultures were grown at either 22 or 37°C. Thestarting density was an A600 of �0.2, and cells were collected when the densityreached an A600 of �2 (about 12 h at 37°C and about 36 h at 22°C). Aliquots (0.5ml) of the culture were collected by centrifugation, resuspended in 40 �l of bufferB (10 mM HEPES-KOH [pH 8.0], 15 mM KCl, 5 mM EDTA), and frozen at�75°C. Aliquots were thawed on ice, and the total volume was brought to 200 mlwith buffer B. Cold glass beads were added to the meniscus. Forty microliters of100% trichloroacetic acid (TCA) was added, and the samples were vortexed attop speed for 4 min. Samples were placed immediately on ice, and 1 ml ofice-cold 5% TCA was added. Samples were spun for 20 min at 14,000 � g in the

cold. The liquid was aspirated, and the pellet was resuspended in 1 ml of coldwater. Samples were spun for 10 min as before, and the supernatant was aspi-rated. Proteins were extracted by resuspending the pellet in 150 �l of samplebuffer (6% sodium dodecyl sulfate [SDS], 0.5 M Tris base) and incubating thesuspension at 50°C for 10 min. Samples were spun for 1 min (14,000 � g), and thesupernatant was removed to a fresh tube. The extraction process was repeated,and the supernatants were pooled. One-third volume of a solution of 0.25 Mdithiothreitol, 50% glycerol, and 0.2% bromphenol blue was added, and thesamples were spun (14,000 � g) for 3 min. The supernatant was transferred to afresh tube.

VLP preparation. Cells were grown and lysed as described previously (12).Extract (7.5 ml) was loaded onto a 70/30/20 (5-/5-/15-ml) step gradient andcentrifuged for 180 min at 28,000 rpm in a Sorvall AH629 swinging-bucket rotor.The remaining extract was saved for use as whole-cell extract. Fractions werecollected by puncturing the bottom of the tube and collecting 1.2-ml fractions. Topellet VLPs, peak fractions 4, 5, and 6 were pooled, diluted to 11 ml with bufferB, and pelleted for 1 h at 35,000 rpm in a Sorvall A1256 fixed-angle rotor. Thepellet was resuspended in 150 �l of buffer B.

Immunoblotting. Whole-cell extracts and purified VLPs were mixed with anequal volume of 2� sample loading buffer (20% [vol/vol] glycerol, 0.125 MTris-Cl [pH 6.8], 5% [wt/vol] SDS, 10% [vol/vol] 14 M �-mercaptoethanol, 0.2%[wt/vol] bromphenol blue) and boiled (3 min) prior to loading on 10 (Gag blots)or 7.5% (Pol blots) SDS gel. Gels were transferred to nitrocellulose (for Gagblots) or a polyvinylidene difluoride (PVDF) membrane (for Pol blots) in Tris-glycine buffer containing 10% methanol at 24 V for 1 h. Membranes wereblocked in phosphate-buffered saline containing 5% nonfat dried milk. Mem-branes were then probed with antibodies as described previously (21). Antibodybinding was detected with the appropriate secondary antibody followed by ECL(nitrocellulose) or ECL-Plus (PVDF) reagent and exposure to X-ray film. Anti-Gag (anti-VLP polyclonal serum R2-F) and anti-IN (monoclonal antibody 8B-11) are described elsewhere (12, 23).

Processing in E. coli. A Ty1 Gag-PR construct (PPR) was prepared as de-scribed previously (19). Briefly, this construct contains a methionine codonpreceding codon 348 of Ty1 Gag and a TAG codon after codon 182 of PR. Theframeshift signal was removed via a single nucleotide deletion to permit expres-sion in E. coli. Cells were lysed in 5 volumes of 1.2� Laemmli buffer. The lysatewas centrifuged at 14,000 � g at room temperature, and the supernatant wastransferred to a fresh tube. The samples were resolved by SDS–14% polyacryl-amide gel electrophoresis and transferred to a PVDF membrane at 300 mA for2 h. Membranes were probed with rabbit polyclonal antibody JH695 as describedpreviously (19).

Biochemical assays. Frameshift assays were done as described previously (27).Plasmids pACTTy and pACTy were a kind gift from M. Cassan. Ty1 cDNAsynthesis assays were performed exactly as described previously (21). RT activityassays were done as described previously (12).

Glucose chase experiments. Galactose induction/glucose chase experimentswere done using strain YH82. For high-temperature induction, cells were inoc-ulated at a density of �8 � 106 to 10 � 106 cells/ml into selective medium (tomaintain the Ty1 plasmid) containing 1% raffinose and incubated at 22°C for 3 h.Cultures were then shifted to 37°C for 2 h prior to induction. Galactose wasadded to 2% final concentration, and cultures were incubated overnight. Glucosewas then added to 2% final concentration to stop induction. The culture wasdivided in half and incubated at 37 or 30°C, with 5-ml aliquots being removed at

FIG. 1. Ty1 processing sites. Translation of the Ty1 element yieldsGag-p49 and Gag-Pol-p199 protein products, which are subsequentlyprocessed by PR. The Gag/PR processing site is present in both trans-lational products. This site is cleaved first in the Gag-Pol-p199 polypro-tein, followed by cleavage at the two downstream sites (21).

TABLE 1. Yeast strains and Ty1 plasmids

Strain Genotype Ty1 plasmida (reference) Ty1 marker Strain background

YH8 MAT� ura3-167 his3200 leu21 trp11 GRF167YH50 MATa ura3-167 his3200 leu21 trp11 spt3-202 S288CYH51 MATa his4-539 lys2-801 ura3-52 spt3-202 pJEF1105 (6) neo S288CYH82 MATa his4-539 lys2-801 ura3-52 trp163 leu21 pJEF724 (3) None S288CJKc1015 MAT� ura3-167 his3200 leu21 pGTy1H3m his3AI (10) his3AI GRF167JKc1031 MATa his4-539 lys2-801 ura3-52 spt3-202 pJEF1105 (6) neo S288CJKc1032 MATa his4-539 lys2-801 ura3-52 spt3-202 pJEF1105PR- (6) neo S288CJKc1054 MAT� ura3-167 his3200 leu21 pJEF1105 (6) neo GRF167JKc1055 MAT� ura3-167 his3200 leu21 pJEF1105PR- (6) neo GRF167W3031-B MAT� ade2-1 can1-100 his3-11,15 leu2-3 trp1-1 ura3-1 pGTy1H3m his3AI (10) his3AI W303Hansen BY4741 MATa his31 leu20 ura30 met150 pGTy1H3m his3AI (10) his3AI S288CYPH652 MAT� ura3-52 his3200 leu21 pGTy1H3m his3AI (10) his3AI YPH (S288C)

a All Ty1 plasmids are GAL-Ty1-H3 URA3 2�m.

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each time point. Cells from each aliquot were pelleted and frozen prior toprotein isolation as described previously (22).

RESULTS

Transposition is temperature sensitive. Ty1 transposition istemperature sensitive. Transposition of endogenous Ty1 ele-ments is sharply reduced at 30°C relative to that at 22°C and isundetectable at 37°C (5, 26). We quantitated Gal-inducedtransposition of a Ty1 element, marked with the his3AI re-porter gene, in 2°C increments from 22 to 36°C in several strainbackgrounds. Transposition frequency is highest in moststrains at �24°C (Fig. 2). Similar to what was found forgenomic elements, the frequency of galactose-induced trans-position decreases steadily as temperature increases, droppingsharply above 32°C. Similar results are observed when a Ty1element containing an uninterrupted reporter gene is used(data not shown). All strains show greater-than-1,000-fold re-duction in transposition frequency from 22 to 36°C. Transpo-sition at 36°C is undetectable in most strains.

Ty1 PR is temperature sensitive. During studies of the Ty1PR protein, Ty1 PR was cloned and heterologously expressedin E. coli (19). The Ty1 PR expression construct used containsan ATG codon immediately upstream of the codon for aminoacid 348 of the Ty1 Gag protein, followed in frame by thecoding sequence for Ty1 PR. The frameshift signal was“erased” by a single base pair deletion that does not alterprotein coding with respect to Gag or PR. The coding se-quence for a carboxy-terminal hexahistidine tag was affixed,followed by a termination codon. Autoprocessing of the fusionprotein yields an �28-kDa PR protein. Immunoblot analysis ofthe purified protein probed with an anti-Gag polyclonal anti-body shows that autoprocessing occurs efficiently at 22 and26°C and to a lesser extent at 30°C and is abolished at 37°C(Fig. 3). The fusion protein remains soluble irrespective of thetemperature at which the cells were grown. As a control, aconstruct bearing an inactivating mutation in the PR active sitewas also expressed. This construct is not processed at anytemperature examined, indicating that the processing observed

in the wild-type construct is not the result of an adventitious E.coli protease. The Ty1 PR is therefore inactive with respect tothe Gag-PR cleavage site at high temperature when expressedin E. coli as a Gag-PR fusion. Although the context of the Gagcleavage site in this construct is likely quite different than thatfor the native Ty1 Gag or Gag-Pol protein, the effect of tem-perature on PR cleavage is severe. This result led us to furtherinvestigate Ty1 PR activity at high temperatures in yeast.

Processing of Pol, but not Gag, is defective at high temper-ature. Since PR activity is apparently innately temperaturesensitive, we examined the processing of Ty1 proteins at vari-ous temperatures in yeast strains containing a Ty1 elementtranscriptionally regulated by the GAL1 promoter. A PR�

mutant version of this Ty1 element was used in parallel toassess nonspecific Ty1 protein degradation at high tempera-ture. Cells were induced to express Ty1 by adding galactose toactively growing cells at 22 or 37°C. A polyclonal antiserum(R2-F) that detects both precursor Gag-p49 and processedGag-p45 was used (21) to probe homogenates prepared fromcells induced at either 22 or 37°C (Fig. 4A). The levels ofGag-p49 processing at 22°C vary by strain origin (Fig. 4A, lane1 versus 5). At 37°C, processing is slightly inhibited in aGRF167 strain. However, in an S288C strain, although thetotal protein yield is less at 37°C, processing is not affected, asthe ratio of p45/p49 does not change (Fig. 4A, lane 5 versus 7).This is in stark contrast to the processing of the Gag-PR con-struct in E. coli, where no processing was detected at 37°C. Theprocessing of Gag-p49 in yeast at high temperature is not dueto nonspecific activation of cellular proteases, as no processingof Gag-p49 is seen using the PR� construct.

Anti-IN monoclonal antibody 8B-11 detects processed IN(Pol-p71; apparent molecular mass, �90 kDa) as well as thefull-length Gag-Pol-p199 polyprotein and any processing inter-mediates containing the IN epitope. 8B-11 was used to assessefficiency of Pol processing. Immunoblots of whole-cell ho-mogenates prepared from cells induced at either 22 or 37°Cwere probed with an anti-IN antibody (Fig. 4B). While pro-cessed IN is readily detected in cells grown at 22°C, it is un-detectable in cells grown at 37°C. Gag-Pol-p199 polyproteinyield is markedly reduced, but detectable, at 37°C. Thus, totalcellular Gag-Pol-p199 levels are reduced and proteolysis of theTy1 Pol polyprotein appears to be defective at high tempera-

FIG. 2. Transposition is temperature sensitive. Several wild-typeyeast strains containing a galactose-inducible Ty1 element on plasmidpGTy1m his3AI were quantitated for transposition following galactoseinduction at the indicated temperatures (as described in Materials andMethods). Strains are as follows: JKc1015 (open circles), W3031-B(solid squares), Hansen BY4741 (solid circles), and PH652 (solid tri-angles). Full strain genotypes are given in Table 1.

FIG. 3. Ty1 PR expressed in E. coli is temperature sensitive. A fusionprotein (PPRHis6) containing the Gag/PR cleavage site followed by theTy1 PR and six histidines was expressed in E. coli. Immunoblots of cultureextracts were probed with polyclonal antibody JH695 to Gag. The fusionprotein undergoes autoprocessing in cells grown at 22, 26, or 30°C but notin cells grown at 37°C. In the PPR�His6 construct, the PR active site ismutated, abolishing autoprocessing.

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ture. As seen with Gag-p49, the processing efficiency of Gag-Pol-p199 at 22°C varies by yeast strain (Fig. 4B, lane 1 versus5). This result suggests that there is a “context sensitivity” forthe processing of the Gag-PR cleavage site. Since Gag-PRcleavage of the Gag-Pol-p199 polyprotein is essential for fur-ther processing, we propose that, at high temperature, Gag-Polis not processed at the Gag-PR site but that Gag-p49 is pro-cessed to some extent.

The lack of visible processed IN at 37°C (Fig. 4B, lanes 3 and7) could alternatively be explained by the marked reduction inGag-Pol polyprotein in these whole-cell extracts. Thus, wefurther investigated production and processing of Ty1 proteinsby immunoblotting purified VLPs. Cell cultures of GRF167origin were induced by galactose at 22 and 37°C, and cellextracts were subsequently fractionated on a sucrose gradient.Peak fractions were pelleted to concentrate and purify VLPs.VLPs are concentrated by this fractionation, indicating thatstable VLPs are formed at high temperature, albeit at reducedyield. Immunoblot analysis of purified VLPs shows an approx-imately threefold reduction in the overall level of Ty1 Gagprotein produced at 37°C (Fig. 4C). The reduction of the Polprotein product is greater, as shown by immunoblots of puri-fied VLPs probed with an anti-IN antibody (Fig. 4D). There is,however, a sufficient Gag-Pol-p199 protein signal present inboth cell extracts and VLPs induced at 37°C to detect pro-cessed IN, if it had been present (Fig. 4D, lane 2 versus 4 andlane 5 versus 10). The reduction in protein levels at high

temperature is significant but far less than the concomitant1,000-fold reduction in transposition frequency.

Transcriptional and translational effects are not significantcontributors to the temperature sensitivity of transposition.The levels of steady-state Ty1 mRNA for cells grown in galac-tose at 22 and 37°C were found to be the same (data notshown). As Ty1 mRNA is very abundant in both conditions, itis unlikely to be a limiting component. The total amount ofGag protein detected in VLPs when cells are grown at 37°C isreduced by approximately threefold compared to that whencells are grown at 22°C. However, the difference in Pol proteinproduction appears to be much greater. Thus, although trans-lation initiation efficiency and stability of Ty1 proteins at hightemperature are unlikely to contribute significantly to reducedtransposition, the difference in the relative amounts of Gagand Gag-Pol could, in principle, result from an effect of tem-perature on frameshifting. A �1 frameshift signal near thecoding sequence for the C terminus of Gag regulates the rel-ative amounts of Gag and Gag-Pol proteins. An AGG codonnear the 3� end of the Gag coding sequence requires decodingby a rare tRNA, frequently resulting in a ribosomal stall (2, 7,31). A �1 frameshift occurs �10% of the time while the stalledribosome awaits a tRNA to decode the AGG codon, producingthe Gag-Pol fusion protein. A temperature-sensitive effect onframeshifting could significantly affect the ratios of Ty1 proteinproducts. Retrotransposition is blocked when the Gag-to-Gag-Pol ratio is disturbed by (i) alteration of expression levels of a

FIG. 4. Processing of Ty Pol proteins is temperature sensitive. (A) Immunoblots of cellular homogenates from yeast cultures induced by galactoseat 22 (10 �l/lane) or 37°C (20 �l/lane) and probed with anti-Gag. The Gag-p49 and processed-Gag-p45 bands each appear as doublets. Two yeast strainsof differing origins, each harboring a galactose-inducible element with either a functional (PR�) or a mutant (PR�) PR, were used. (B) Same as panelA, except that immunoblots were probed with anti-IN. Gag-Pol-p199 and IN (Pol-p71) are indicated. Homogenate from cells induced at 22 (20 �l/lane)or 37°C (40 �l/lane) was loaded onto the gel. (C) Immunoblot of whole-cell extracts and purified VLPs from a GRF167 strain (JKc1015) induced at 22or 37°C and probed with anti-Gag. The numbers above the lanes indicate the volumes of original sample loaded. A fraction above a lane indicates thedilution of VLPs loaded. (D) Immunoblot of whole-cell extracts and purified VLPs from a GRF167 strain (JKc1015) induced at 22 or 37°C and probedwith anti-IN. The numbers above the lanes indicate the volume (in microliters) of original sample loaded.

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single-copy tRNA gene that inhibits frameshifting or (ii) ex-pression, in trans, of a protein known to inhibit �1 ribosomalframeshifting (11, 15, 28, 31).

Frameshifting efficiency was investigated by using a con-struct containing the �-galactosidase open reading frame(ORF), followed by the Ty1 frameshift signal and the lucif-erase ORF (27). A control construct encodes luciferase as anin-frame fusion to �-galactosidase (Fig. 5). A successful �1frameshift in the first construct thus results in translation of theluciferase reporter protein. Levels of luciferase activity wereassayed and standardized to �-galactosidase activity. The re-sults (Fig. 5) show that frameshifting in the context of thepACTy construct at 22°C is strain dependent and is, at best,fourfold more efficient than the 10% frameshifting seen in thecontext of a Ty1 element mRNA. The relative change in theluciferase-to-�-galactosidase activity ratios at 37°C implies anincrease in frameshifting efficiency that approaches or exceeds100% at high temperature. However, it is notable that �-ga-lactosidase activity in the context of pACTy is reduced bytwofold at high temperature, thereby artifactually increasingthe apparent frameshift efficiency. Additionally, we cannot ruleout effects of differences in enzyme stability at high tempera-tures, as luciferase activity is notably reduced in both con-structs at 37°C. However, an increase in frameshifting effi-ciency would be expected to result in increased readthroughtranslation and a greater amount of Gag-Pol protein than Gagproduced at 37°C. Our immunoblots of VLPs show a reductionin Gag-Pol protein synthesis compared to that of Gag at 37°C.Thus, the modest increase in frameshifting efficiency is pre-sumably insignificant with respect to Ty1 transposition.

cDNA synthesis is defective. Since the cleavage of the Gag-Pol-p199 protein was inhibited, we examined how this affectedfurther steps in the Ty1 life cycle. Cleavage of the Gag-Pol-p199 sites is a semiordered process in that Gag/PR cleavagemust occur first, but the subsequent cleavages do not appear to

be ordered. Mutational analysis of the cleavage sites has shownthat cleavage of PR/IN and IN/RT sites is not required forcDNA synthesis but that cleavage of the Gag/PR site is re-quired. We therefore examined Ty1 cDNA synthesis by South-ern blotting total DNA isolated from cell cultures grown atdifferent temperatures (Fig. 6). No Ty1 cDNA is detectable inSouthern blots of DNA isolated at 36°C. Similarly, VLPs iso-lated from cells grown at 36°C do not contain any cDNA. Theamount of cDNA detected at 30°C is �20% of the amountseen at 22°C, consistent with the levels of transposition ob-served (Fig. 2). The outcome of the reverse transcription pro-cess at high temperature phenocopies that of a PR� element atlow and normal growth temperatures.

FIG. 5. Temperature effects on frameshifting. The diagram shows the constructs used to measure frameshifting. pACTTy contains theluciferase reporter gene in frame with the �-galactosidase reporter gene. In the pACTy construct, the Ty1 frameshift sequence has been insertedbetween the two ORFs. Expression of the luciferase reporter indicates the efficiency of the Ty frameshift site. The reporter gene activity from theconstructs was measured in triplicate in two yeast strains grown at 22 and 37°C. The �-galactosidase (�-gal) and luciferase activities are the averagesof the three measurements, and the standard deviations are in parentheses. Luciferase activity, normalized to �-galactosidase activity, is used tocalculate the percent frameshift efficiency for each strain at both temperatures. Frameshift efficiency is calculated as 100 times the ratio of luciferaseactivity/�-galactosidase activity for the pACTy plasmid divided by the corresponding ratio for the pACTTy plasmid.

FIG. 6. Ty1 cDNA synthesis as a function of temperature. Cells(YH51; Table 1) containing a galactose-inducible Ty1 element weregrown at the indicated temperatures. Southern blot analysis of ex-tracted DNA indicates that production of the 3.5-kbp cDNA productis markedly reduced at 30°C and is undetectable at 37°C. Nucleic acidswere extracted, digested with EcoRI, treated with RNase, electro-phoretically separated (1% agarose gel), and transferred. The mem-brane was probed with a [32P]-labeled neo cDNA probe. The larger�10.0-kbp band corresponds to the Ty1 plasmid.

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RT activity is not inherently temperature sensitive but isreduced in VLPs formed at high temperature. Since no cDNAis detectable, we investigated RT activity in vitro at high tem-perature. VLPs prepared from galactose-induced cultures at22 and 37°C were assayed for in vitro RT activity at both 22 and37°C. Since VLP yield is reduced at high temperature, theamounts of VLPs added to the reaction mixtures were normal-ized to levels of Gag protein in the VLPs (Fig. 4C). As seen inFig. 7, RT activity is not inherently temperature sensitive;VLPs formed at 22°C have the same levels of RT activity at 22and 37°C. However, VLPs formed at 37°C have �15-fold re-duced activity at either temperature. In vitro RT activity canreadily be detected in the context of the full-length polypro-tein, as both PR� active-site mutants and Gag*PR cleavagesite mutants have RT activity on an exogenous primer template(21, 32). Despite the reduction in Gag-Pol polyprotein levels at37°C, there is a significant amount of RT activity above back-ground in these VLPs, suggesting that what little RT is presentstill possesses activity in the in vitro assay.

The PR defect is not reversible. To determine whether theprocessing defect is reversible, yeast cultures were induced bygalactose at 37°C overnight, after which glucose was added tohalt Ty1 gene expression. Cell aliquots were further incubatedat 37°C or shifted to 30°C at various intervals. Immunoblots oftotal cell protein from aliquots were probed with an anti-INantibody. As shown in Fig. 8, IN was not processed from thepolyprotein upon subsequent incubation at 30°C. Additionally,the half-lives of the polyprotein at 30 and 37°C are the same, asshown by the similar degradation rates of the Gag-Pol-p199polyprotein. Thus, a shift down to the permissive temperatureafter galactose induction does not recover processing.

DISCUSSION

In this paper we demonstrate that a major block to trans-position at high temperature is the inhibition of PR activity,exacerbated by a decrease in overall Ty1 protein levels. Thedecrease in overall Gag-Pol protein level observed at 37°Ccould be a consequence of the decreased PR activity or it couldbe due to some unrelated change in cellular physiology. Whenexpressed in E. coli, recombinant Ty1 PR is nonfunctional athigh temperature, suggesting an intrinsic temperature sensitiv-ity. In yeast, cleavage of the Gag-Pol-p199 polyprotein is com-pletely blocked at high temperature, and cleavage of the Gagprotein is slightly reduced in some strains. In VLPs formed athigh temperature, exogenous RT activity is significantly re-duced but detectable, presumably reflecting a decrease in totalRT protein, as this exogenous RT activity is not inherentlytemperature sensitive in vitro. Endogenous RT activity, deter-mined by measuring Ty1 cDNA production, decreases withincreasing growth temperature, consistent with reduced RTamounts and activity, and is undetectable at 37°C. Thus, theexogenous RT activity detected in purified VLPs is insufficientto produce detectable cDNA in vivo. This conclusion agreeswith earlier studies in which PR-deficient mutants failed tosynthesize cDNA (21, 32). We hypothesize that a temperature-induced conformational change in the Ty1 Gag-Pol polypro-tein reduces the activity of both PR and RT, resulting in nearlyundetectable levels of transposition. It is likely that the reduc-tion in Ty1 protein levels at 37°C also contributes to the severetransposition defect, as Ty1 transposition does not necessarilycorrelate with the amount of detected protein. Curcio andGarfinkel previously showed that expression of a galactose-inducible Ty1 element increases transposition efficiency at theposttranslational level, and their studies suggested that theactivity of endogenous Ty1 elements may be limited by thelevels of PR activity (9). Correspondingly, we have shown thata major defect in high-temperature transposition is posttrans-lational. It is possible that a limiting factor for transpositionfalls below a threshold level at temperatures above 32°C, con-tributing to the rapid drop-off in transposition.

FIG. 7. RT activity is not temperature sensitive. VLPs were purifiedfrom cells (JKc1015; Table 1) induced with galactose at 22 or 37°C. RTactivity using exogenously added primers and templates was measured at22 and 37°C for both VLP preparations. Assay samples were normalizedto the Gag protein (Fig. 4C) by adding a 3.5-fold-greater volume of the37°C VLP preparation. N/A (none added), background level of incorpo-rated radioactivity when buffer B is added in place of VLPs.

FIG. 8. The PR defect is not reversible. Cells (YH82; Table 1) wereinduced with galactose overnight at 37°C. Following addition of glu-cose, the cultures were shifted down to 30°C or maintained at 37°C.Numbers indicate the time points, in hours, at which aliquots wereremoved. Total cell protein was analyzed by immunoblotting using anantibody to IN. No processed IN was detected.

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A recently published study determined the necessity of eachof the Ty1 cleavage sites for Ty1 transposition (21). Proteolyticprocessing of the three Ty1 cleavage sites occurs in a regulatedand semiordered manner. Gag/PR must be cleaved first, afterwhich cleavage of the other two sites occurs without requiredorder. Blockage of either of the cleavage sites in Pol (PR/INand IN/RT), independently or in combination, did not affectcDNA production in these mutants, and the affected site(s)shows a cleavage defect. However, blocking the Gag*PR cleav-age site abolished cleavage at all three sites and also blockedcDNA production. A PR� active-site mutant, in which noprocessing occurs, was also defective in cDNA synthesis. Wefound that, in VLPs formed at high temperature, a differentphenotype was observed. Processing at the two Pol sites(PR/IN and IN/RT) was completely abolished. However, un-like what was found for PR� and Gag*PR processing sitemutants, processing at high temperature in yeast was not com-pletely blocked, as indicated by the presence of processedGag-p45 protein from the Gag-p49 primary translation prod-uct. This processing of p49 required active Ty1 PR. Takentogether, these results point to a context-dependent decreasein PR activity wherein Gag-Pol is not processed at the Gag-PRjunction but Gag-p49 is processed. Curiously, both of theseprocessing events occur at the same scissile bond located be-tween histidine 401 and asparagine 402 (Fig. 1).

Since we observe processing of Gag, it appears that VLPsformed at high temperature harbor an additional defect, otherthan processing, that blocks cDNA production. Production ofcDNA is gradually reduced as temperature increases. A mildincrease in temperature (to 30°C) reduces the amount ofcDNA produced, although Gag-Pol-p199 processing occursreadily at this temperature. Thus, as temperature increases,VLPs become less efficient at producing cDNA. The observedreduction of RT activity was still expected to produce a smallbut detectable amount of residual cDNA synthesis at 37°C.However, we failed to detect any cDNA. The cDNA synthesisdefect might therefore be exacerbated by the slight decrease inGag processing. In summary, it is likely that the temperaturesensitivity of Ty1 retrotransposition reflects the combined ef-fects of deficiencies in both proteolytic processing and in vivocDNA synthesis.

Previous studies have shown that PR� mutants are defectivein cDNA production, not because RT is inactive but because ofa defect in accessing the endogenous primer or template or adefect in forming dimeric RNA or both (13, 21, 32). The initialstep to cDNA synthesis in Ty1 is the annealing of the host-encoded tRNAi

Met primer to the primer-binding site (PBS) onthe Ty RNA. Data from studies of PBS region mutants suggestthat formation of the primer/template complex is a tempera-ture-sensitive step in transposition (18). A G:U mismatch in-troduced into the primer/template complex reduces transposi-tion activity to approximately one-third wild-type levels at27°C. Conversely, extension of the PBS complementarity re-gion from 10 to 12 nt conferred a slight temperature resistanceto transposition (17). At 34°C, transposition levels of an ele-ment containing a 12-nt PBS are nearly 100-fold greater thanthat of a wild-type element. However, transposition in thesemutants is still substantially less than that seen at 22°C. There-fore, the lack of cDNA at high temperature may be attributedonly in part to reduced efficiency of primer/template forma-

tion. It is also possible that temperature-induced conforma-tional changes in the template/primer complexes that form athigh temperature further reduce the efficiency of RT initiation.

Transposition is very sensitive to the conformation of theprimer/template complex. Introduction of a G·U mismatch,while not expected to significantly affect primer/template an-nealing, does result in a slightly temperature-sensitive pheno-type. Additionally, a single mismatch in the primer/templatecomplex reduces transposition dramatically (16). Thus, tem-perature may have an effect on priming at two steps: hightemperature may reduce the efficiency of primer/template an-nealing and may affect the conformational structure of com-plex that forms. Recent studies suggest that the amino termi-nus of the Ty1 PR plays an important role in the initiation ofreverse transcription in vivo and that Gag is involved in primer-template annealing (8, 20).

Primer/template formation, the initial step in cDNA forma-tion, is followed by RT-mediated DNA synthesis. The forma-tion of cDNA in VLPs is a measure of the endogenous activityof RT. In this study, we also measured the ability of RT tosynthesize DNA from an exogenous primer/template complex.Formation of this exogenous oligo(dG)/poly(C) primer/tem-plate complex is not temperature dependent. Our data showthat RT activity is not inherently temperature sensitive. RTactivities at 22 and 37°C in VLPs formed at 22°C are compa-rable. However, RT activity is greatly reduced in VLPs formedat high temperature, regardless of whether the in vitro assay isperformed at 22 or 37°C. PR� mutant VLPs prepared at 22°Chave readily detectable RT activity in these assays, indicatingthat processing is not required for activity and that RT isfunctionally active as part of the Gag-Pol-p199 polyprotein(21). These results argue that high temperature affects theconformation of the RT protein attained during synthesis, ren-dering it inactive. That the RT activity is not inherently tem-perature sensitive suggests that RT folding occurs during par-ticle formation as part of the polyprotein and that RT, oncefolded, remains stable. Thus, particle formation at high tem-perature could result in an inactive conformation. Folding islikely to be a complex process, affected by interactions withother Ty1 or host factors (29). An alternative hypothesis is thatexogenous RT activity is reduced simply because there is mark-edly less RT protein in VLPs formed at high temperature. Wenormalized to Gag proteins in the exogenous RT assays, andactivity was still reduced 15-fold. Therefore, the inactivation ofRT is significant.

The Gag-Pol-p199 protein is readily detectable at 37°C, butthe overall levels of Pol protein products are reduced relativeto levels seen at 22°C. Our immunoblot analyses suggest thatthe reduction of total Pol protein products at 37°C is greaterthan the reduction in Gag protein levels. We tested the effectof temperature on frameshifting and found that frameshiftingis modestly affected, being approximately twofold more effi-cient at 37°C than at 22°C. A more efficient frameshiftingmechanism would be expected to yield relatively more Polprotein product, not less. However, our findings resemble theresults of a previous study on the effects of increased frame-shifting efficiency. Frameshifting is mediated by a ribosomalstalling event caused by a rare tRNAArg codon at the �1frameshifting site. Deletion of the gene for this tRNA in-creases frameshifting 3- to 17-fold (15). Interestingly, this mu-

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tation did not result in an accumulation of Pol products; ratherthe particles displayed a processing defect in that processed INis undetectable in the mutant strain. As for VLPs made at hightemperature, the processing of Gag in this mutant was notaffected. Thus, an increase in frameshifting results in a pro-cessing defect, perhaps due to reduced PR activity in the con-text of particles forming in the presence of skewed Gag/Gag-Pol protein ratios. It therefore remains possible that a slightincrease in frameshifting efficiency at high temperature couldcontribute to the observed Pol processing defect.

We have shown that the temperature sensitivity of transpo-sition is due to a reduction in both PR and RT activity, result-ing in a profound processing defect and a lack of any detect-able cDNA in VLPs formed at 37°C. We hypothesize thataberrant folding at high temperature adversely affects the ac-tivities of both of these enzymes. Additionally, reduced proteinlevels, primer/template formation, and frameshifting effectsmay contribute to the phenotype of high-temperature VLPs. Itis unknown whether the activity of IN is also affected by hightemperature in vivo. Although reduced PR cleavage and RTfunction are major blocks to transposition at high temperature,the complete inhibition may well be due to the additive effectsof defects in other steps as well. Such a complex control systemallows adjustment to multiple environmental signals. Host mu-tants that partially restore transposition and processing at hightemperature have been isolated (J. B. Keeney, unpublisheddata). Further characterization of these mutants will identifyadditional host cell-mediated events in the complex control oftransposition.

ACKNOWLEDGMENTS

This work was supported in part by NIH grants GM36481 to J.D.B.and GM54291–02 to J.B.K. and grants from The William J. von LiebigFoundation and the Howard Hughes Medical Institute (no. 52002813)to Juniata College.

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