Ala-scanning mutagenesis strategy.
The Ty3 NC domain spans carboxyl-terminal residues 234 through 290 of Gag3 (Fig. ). (Residues are numbered according to the Gag3 precursor.) In order to characterize the contribution of the NC domain to the Ty3 life cycle, a set of 17 mutants was created (Table ). The mutations included seven Ala-scanning mutations in which windows of five residues containing two or more charged residues were substituted at charged positions with Ala; four single and two double Ala substitutions of conserved positions within the zinc-binding domain; and two deletion mutants, one lacking all but two residues of the amino-terminal domain (NTD) of NC, including 10 of 17 basic residues (aa 236 to 265, ΔNTD), and the other lacking 45 of 57 residues in NC (aa 237 to 281, ΔNC) (Fig. ). Deletion mutants were designed to retain PR processing and Gag3-Pol3 frameshift sites. Mutants were characterized by using transposition, immunoblot, sedimentation, packaging, replication, and localization assays.
Summary of Ty3 NC mutant phenotypes
Retrotransposition of Ala-scanning mutants.
A qualitative genetic assay of integration into a plasmid containing a tRNA gene target was performed to determine the effects of NC mutations on retrotransposition frequency (see Materials and Methods). Two independent transformants were tested for wt Ty3 and each mutant at 24 and 30°C. The transposition properties of these mutants are summarized in Table .
Five of the seven Ala-scanning mutants (R236A/R238A/R239A, R249A/R251A/R252A, K263A/R265A, E279A/R281A, and R283A/K284A) had transposition frequencies similar to that of the wt at both temperatures (Table ). Two mutants were affected by temperature; one was severely defective at 30°C (K271A/K272A/E273A), and one was modestly affected at 24°C (R258A/E259A/E260A). Single zinc-binding motif mutants (C267A, C270A, H275A, and C280A) were each severely blocked for transposition at both temperatures. Thus, transposition appeared relatively resistant to small changes in charged residues but was sensitive to any change in the conserved zinc-binding motif.
Precursor polyprotein maturation.
Ty3 PR processing was used as an indirect measure of proteins that were competent to assemble in a correctly folded state. YTM443 transformants carrying either pDLC201 with wt Ty3 or mutant derivatives were grown in SR-Ura medium and induced to express Ty3 for 24 h as described in Materials and Methods. WCEs were prepared, and the processing of wt and mutant Gag3 and Gag3-Pol3 was analyzed by immunoblot analysis with anti-CA, anti-NC, and anti-IN antibodies (Fig. and Table ).
FIG. 2. Mutations in NC affect Ty3 Gag3 and Gag3-Pol3 protein processing. Cultures were induced for Ty3 expression for 24 h at 24°C, and WCEs were prepared under denaturing conditions and processed for immunoblot analysis as described in Materials and (more ...)
Extracts from cells expressing wt Ty3 analyzed with anti-CA showed Gag3, p27, and CA species (Fig. ). Analysis of extracts with anti-IN showed a predominant band consistent with the migration of the 61-kDa IN (Fig. ). The IN antibody also detected the slower mobility Gag3-Pol3 species (33
; data not shown).
Extracts of cells expressing wt Ty3 and each of the five mutants with wt levels of transposition (R236A/R238A/R239A, R249/R251A/R252A, K263A/R265A, E279A/R281A, and R283A/K284A), as well as the mutant with modest temperature sensitivity (R258A/E259A/E260A), showed similar amounts of Gag3 and p27 and a significantly greater amount of CA than any other species (Fig. ). The amounts of NC and IN were comparable to that produced by wt Ty3 (Fig. ). The temperature-sensitive mutant K271A/K272A/E273A showed a modest reduction in Gag3 processing under permissive conditions (Fig. ) but the wt levels of IN (Fig. ).
All of the zinc-binding motif mutants, (C267A, C270A, H275A, C280A, C267A/C280A, and C270A/C280A) displayed reduced and aberrant processing of Gag3 (Fig. ). In immunoblot analyses with the anti-CA antibody, these mutants had high levels of precursor Gag3, and two showed one species migrating slightly more slowly than wt p27. Mutants C267A and C267A/C280A showed no detectable wt Gag3 processing products but did show an unidentified species migrating more slowly than Gag3 (data not shown) and some species migrating more slowly than the NC species. The zinc-binding motif mutants displayed no or barely detectable levels of either of the NC species which were most prominent in cells expressing wt Ty3 (Fig. ). However, they showed increased amounts of two slightly larger species, possibly arising from loss of SP-NC cleavage. Somewhat surprisingly, only one single zinc-binding motif mutant (C267A) displayed significantly reduced amounts of IN, but both double substitution mutants showed reduced amounts (Fig. ). This could have arisen from a reduction in processing or a reduction in the total amount of stable Gag3-Pol3 in cells, since the level of the higher-molecular-weight Gag3-Pol3 species was also reduced compared to that in cells expressing wt Ty3 (data not shown).
The wt or intermediate processing phenotypes of mutants were consistent with transposition results and confirmed that the basic residue replacements did not have significantly deleterious effects. In order to increase the probability that functions of the non-zinc-binding basic residues were disrupted, one additional mutation was created that was a deletion of residues 236 through 265, including a total of 10 of 17 basic amino acids (ΔNTD). Residues 237 to 281 of NC (45 of 57 residues), including the entire zinc binding motif (ΔNC) were deleted in another construct. Both of these mutants showed unprocessed, truncated Gag3, but also significant amounts of what appeared to be mature CA (Fig. ). As expected, because the anti-NC antibody was raised against a peptide representing the NTD, NC products were undetectable for the ΔNTD and the ΔNC mutants. In order to verify that the Gag3 processing pattern was attributable to Ty3 PR and therefore implied assembly, a second mutation was introduced into both deletion mutants to change the PR active site Asp to Ile. This mutation was previously shown to inactivate PR (31
). In both cases, introduction of this mutation resulted in full-length Gag3, indicating that the mutants displayed bona fide PR processing activity.
Particle formation by Ty3 NC mutants.
The processing pattern of many of the mutants indicated that protein multimerization or assembly was occurring in some, but possibly not all, mutants. In order to better understand the point at which transposition was disrupted, we next investigated the ability of individual mutants to form sedimentable species. A subset of mutants representing ones with wt transposition frequency (R236A/R238A/R239A) and those affected for transposition (K271A/K272A/E273A, C267A, C270A, H275A, and C280A), as well as double mutants (C267A/C280A and C270A/C280A), were tested. In order to maximize the possibility that mutant multimerization could be distinguished from wt, cells were induced for Ty3 expression for only 6 h at 24°C. Protein was extracted under nondenaturing conditions as described in Materials and Methods.
WCEs of cells expressing wt Ty3 had Gag3, p27, and CA in the pellet fraction in the absence or presence of detergent (Fig. ). In the presence of detergent, some Gag3, but predominantly p27 and CA, were solubilized. This suggested that Gag3 and its processed species were found in different types of complexes, some of which either were associated with membranes or were more labile in the presence of detergent. For the NC mutants tested, all WCEs contained detergent-insoluble, pelletable material. This finding indicated that, overall, multimerization was robust (Fig. ). Mutants that displayed substantial processing (R236A/R238A/R239A, K271A/K272A/E273A, and C280A) showed pelleting patterns similar to that of the wt. Consistent with the observation that predominantly processed Gag3 species were solubilized by detergent, the remaining mutants with little or no Gag3 processing did not release Gag3 species into the supernatant. These results indicated that mutations within hydrophilic patches or even within the zinc-binding motif in the NC domain did not significantly decrease the ability of Gag3 proteins to form complexes but did affect the nature of the Gag3-Gag3 interaction so that processing did not occur.
FIG. 3. Sedimentation analysis of NC mutants. Cells were induced for 6 h at 24°C, and WCEs were prepared in native conditions and incubated with or without NP-40 as described in Materials and Methods. Samples were fractionated by electrophoresis on a (more ...) Protection of genomic RNA.
A subset of mutants displayed significant processing but were nonetheless profoundly defective in transposition. In order to better understand the basis of the transposition defect, the ability of particles to protect RNA was investigated (Fig. and Table ). wt Ty3 and Ty3 PR−
(immature) VLPs have been previously shown to protect Ty3 RNA from benzonase digestion (33
; unpublished data), so the inability to process alone does not cause a defect in RNA packaging.
FIG. 4. NC mutations reduce packaging of genomic RNA. Cultures were induced for Ty3 expression for 24h at 24°C, and WCEs were prepared under native conditions as described in Materials and Methods. Positive control in vitro-transcribed Ty3 RNA (Ty3 runoff (more ...)
Cells expressing wt Ty3, two temperature-sensitive phenotype mutants (R258A/E259A/E260A and K271A/K272A/E273A), the six mutants with zinc-binding domain substitutions (C267A, C270A, H275A, C280A, C267A/C280A, C270A/C280A), and the two deletion mutants (ΔNTD and ΔNC) were examined. Cells were induced to express Ty3 for 24 h at 24°C. WCEs were prepared under nondenaturing conditions and incubated in the presence or absence of benzonase as described in Materials and Methods. In vitro-generated, truncated Ty3 transcripts were added to all reactions to monitor completion of benzonase digestion. Nucleic acid in these extracts was analyzed by Northern blotting using a Ty3-specific probe. Under these conditions, 33% (±8%) of the wt Ty3 RNA was protected from benzonase digestion compared to control, untreated extracts incubated on ice. The in vitro Ty3 transcripts were completely digested by benzonase treatment (Fig. ).
The Ala-scanning mutants that transposed at wt frequencies at the permissive temperature (R258A/E259A/E260A) showed protection of RNA similar to wt Ty3 (Fig. ). Mutant K271A/K272A/E273A had decreased transposition and Gag3 processing. Surprisingly, it had 43% protection of RNA. Substitution of only one zinc-binding motif residue (C267A, C270A, H275A, and C280A) caused decreased protection, resulting in levels of RNA protection ranging from 10 to 13%. These results suggested that incomplete protection of RNA was achieved even in the presence of a compromised zinc-binding motif structure but clearly implicated the zinc-binding motif in the packaging of genomic RNA. Mutant C267A, which showed no Gag3 processing (Fig. ), was not distinguished from other mutants by this criterion. Both double zinc-binding motif substitution mutants (C267A/C280A and C270A/C280A) and ΔNTD, which retained the zinc-binding motif, as well as ΔNC, showed lower levels of extractable RNA, suggesting that there was less protection of RNA.
As discussed above, NC acts both in RNA genome packaging and as a chaperone in primer annealing. Analysis of RNA packaging indicated that at least one mutant (K271A/K272A/E273A) competent to protect RNA was deficient for transposition. Thus, we investigated the level of replicated extrachromosomal Ty3 cDNA in cells induced for Ty3 expression (Fig. and Table ).
FIG. 5. Mutations in basic residues and zinc-binding motif show different effects on cDNA production. Cultures were induced for Ty3 expression as described in the legend of Fig. and processed for Southern blot analysis as described in Materials (more ...)
Coincident with wt processing phenotypes and near-wt transposition frequency, the basic mutants (R236A/R238A/R239A, R249A/R251A/R252A, R258A/E259A/E260A, K263A/R265A, E279A/R281A, and R283A/K284A) produced cDNA/plasmid ratios in the same range or higher than wt Ty3. Mutant K271A/K272A/E273A, which had temperature-sensitive transposition but only a modest decrease in processing and full RNA protection, had decreased levels of cDNA (Fig. ). All single zinc-binding motif mutants (C267A, C270A, H275A, and C280A) that had reduced and aberrant Gag3 processing and did not produce mature NC had no detectable levels of cDNA (Fig. ). Only mutants that showed transposition had detectable levels of cDNA.
Localization of mutant Ty3-RFP and P bodies.
Our earlier studies showed that Ty3 protein and RNA were both associated with P bodies, leading to the hypothesis that these were sites of assembly. In the present study, we probed the interdependence of Ty3 protein and RNA targeting.
We first tested whether the NC domain was important for Ty3 protein and RNA cluster formation and for colocalization with P bodies. In order to visualize Ty3 mutant proteins in living cells, the NC mutations were introduced into the Ty3-RFP expression vector in which POL3
is fused in frame to the RFP-coding region. wt and mutant Ty3-RFP elements were expressed in a strain that expresses the P-body marker Dhh1-GFP from its native chromosomal locus. In cells not expressing Ty3, there are multiple small Dhh1-GFP foci. However, in cells expressing Ty3 there is typically only one or two P bodies that are enlarged compared to those in wt cells (Fig. ). Ty3-RFP and Dhh1-GFP, as well as other P-body markers, typically colocalize with these enlarged foci (4
FIG. 6. NC function is required for full association of Ty3-RFP and P-body clusters but not for Ty3-RFP cluster formation. wt and NC mutant Ty3-RFP was expressed in a strain with Dhh1-GFP under the native promoter. Mutants shown are representative of two independent (more ...)
Cells were induced for expression of wt Ty3-RFP and nine mutants for 6 h at 24°C prior to examination. Two independent transformants were tested for each mutant. For wt and mutant K271A/K272A/E273A, Ty3-RFP colocalized with Dhh1-GFP for two independent transformants (Fig. and data not shown). Cells expressing the single zinc-binding motif substitution mutants, C267A and C270A and double substitution mutants, C267A/C280A and C270A/C280A, all of which showed reduced Gag3 processing, and RNA protection, generated notably smaller fluorescent clusters. However, Ty3-RFP and Dhh1-GFP fluorescent foci when detectable colocalized (Fig. and data not shown). Cells expressing zinc-binding motif mutants H275A and C280A were similar to the wt in both size of Ty3-RFP clusters and colocalization with Dhh1-GFP.
Cells expressing the ΔNTD mutant had a significant fraction of Ty3-RFP foci with which the P-body marker did not colocalize, and for cells expressing the ΔNC mutant this was the case for the majority of Ty3-RFP foci (Table ). These findings showed that NC is not essential for cluster formation but contributes to Ty3 protein association with P-body components.
Ty3-RFP and Dhh1-GFP localization in cells expressing NC deletion mutants
Localization of Ty3 RNA and P bodies.
We next determined the effect of mutations in the NC domain on localization of Ty3 RNA with P bodies. These experiments were designed similarly to those for localization of mutant Ty3 NC proteins except that Ty3 RNA was marked downstream of POL3
with two copies of the MS2 bacteriophage coat protein binding site (Ty3-MS2) and visualized by interaction with the MS2 phage capsid RNA-binding protein fused to RFP (MS2-RFP) (4
). Cells were induced to express Ty3-MS2 for 6 h at 24°C and were examined by fluorescence microscopy.
As previously reported, MS2-RFP fluorescence was diffuse, and Dhh1-GFP foci were small and scattered in cells not expressing Ty3-MS2 (4
) (Fig. ). Fluorescence imaging of cells expressing wt Ty3-MS2, MS2-RFP, and Dhh1-GFP showed one or two large, bright foci in which RFP and GFP fluorescence colocalized. The basic mutant K271A/K272A/E273A produced a similar pattern. Ty3-MS2 mutants containing substitutions C267A, C270A, and C267A/C280A produced significantly fewer and smaller Ty3-MS2 and Dhh1 foci. The C280A and C270A/C280A mutants also showed reduced numbers and size of foci, but they were more similar to wt and Ty3-MS2 and Dhh1 foci colocalized. Mutant H275A was most similar to the wt (Fig. ). Cells expressing ΔNTD and ΔNC mutants displayed minimal Ty3-MS2 foci, and Dhh1-GFP foci were similar to those in cells not expressing Ty3. Thus, Ty3-MS2 RNA clusters, similar to Ty3-RFP protein clusters, were very sensitive to mutations at positions 267 and 270 of the zinc-binding motif. In cells expressing the ΔNTD and ΔNC mutants, Ty3-MS2 foci were also minimal (Fig. and unpublished data).
FIG. 7. NC function is required for Ty3 RNA cluster formation. NC mutations were introduced to pTy3-MS2 and yeast strain BY4741 expressing Dhh1-GFP was transformed with pTy3-MS2 and pMS2-RFP and induced for 6 h at 24°C. Control cells were not induced. (more ...) Nuclear localization of Gag3 in cells expressing specific NC mutants.
Because our RFP reporter was fused to Gag3-Pol3, we also used immunofluorescence with anti-CA antibodies to directly examine the localization of Gag3 in cells expressing the NC mutants (Fig. and data not shown). Cells were induced to express wt and mutant Ty3 for 6 h at 24°C (4
). As described previously, a majority of cells expressing wt Ty3 showed accumulation of CA in one or two large foci per cell, which were separate from the nuclear DAPI (4′,6′-diamidino-2-phenylindole) fluorescence. In contrast, cells expressing the C267A mutant showed diffuse anti-CA nuclear fluorescence and an irregular nuclear boundary. Cells expressing the other zinc-binding motif mutants, ΔNTD, and ΔNC mutants showed one to three Gag3 foci and some cytoplasmic Gag3. In about one-third of cells, one focus overlapped with DAPI nuclear staining (Fig. and data not shown). In cells where fluorescence indicated the presence of the CA domain, the majority of the nuclei contained diffuse fluorescence. Because the Ty3-RFP reporter did not directly measure Ty3 Gag3-Pol3 species, we also used anti-IN in immunofluorescence experiments to monitor the localization of the IN domain. Although differential sensitivity of the antibodies used did not allow us to conclude that there is no Gag3-Pol3 in the nucleus, IN was detected in patterns similar to that of Ty3-RFP and was not detected in the nuclei of cells expressing the NC mutants (data not shown). Thus, mutations that disrupted Gag3 binding to Ty3 RNA resulted in the nuclear localization of Gag3 but not of Gag3-Pol3.
FIG. 8. Loss of NC function results in nuclear localization of Gag3. NC mutations were introduced to Ty3 in pDLC201, and it was transformed into yeast strain yTM443. Cells were induced for 6 h at 24°C as described in Materials and Methods. Control cells (more ...) EM visualization of particle formation by NC mutants.
The analysis described above indicated that each of the eight mutants that was defective in transposition was competent to form some type of complex. Nonetheless, various degrees of difference from the wt were observed in the patterns of RNA and protein localization and RNA packaging. In addition, it appeared that in some mutants, localization of the Gag3-Pol3 RFP marker and Gag3 differed. In order to better understand the nature of the assembly defects, cells expressing a subset of these mutants were examined by EM. Cells expressing wt Ty3 and its mutant derivatives were induced for 6 h at 24°C and were processed as described previously (33
). Ty3 wt, zinc-binding motif mutants C267A, C270A, C280A, C267A/C280A, and mutants ΔNTD, ΔNC, and ΔNC(PR−
) were examined (Fig. ). One hundred cells were examined for each mutant type. Cells expressing wt Ty3, as previously described (33
), showed large multilobed clusters of particles averaging 40.9 ± 4.0 nm in diameter with electron-dense centers. These correlated well with the multilobed appearance of large fluorescent foci in cells expressing Ty3-RFP. Mutant C267A and mutant C267A/C280A were severely defective in both protein processing, RNA protection, and Ty3 clustering with P bodies. Expressing cells showed accumulation of electron-dense nuclear material. Multiple round patches with poorly defined edges that ranged from 37 to about 80 nm in diameter were also observed in these nuclei. Scattered, more discrete, particle-like forms were observed outside nuclei (Fig. and data not shown). Measurement of seven of these particle-like forms in cells expressing mutant C267A showed that the diameter was close to that of wt Ty3 VLP at 40.2 ± 3.1 nm. However, these lacked the dense interior of wt particles. Despite the occurrence of unusual, apparently membrane-related structures in cells expressing wt Ty3, dense unstructured nuclear aggregates were not observed and nuclear particles similar to Ty3 particles were extremely rare. Cells expressing C270A showed types of molecular defects similar to those of cells expressing C267A and C267A/C280A but were less severely affected. The electron micrographs showed dense aggregates and associated round dense forms in nuclei similar to those in cells expressing C267A. However, cells expressing C270A differed in that the continuum of forms was shifted away from aggregates and unstructured, round patches toward particles that lacked dense centers but did have defined edges. The cytoplasm of a fraction of these cells showed distinct clusters of particles. Mutant C280A was the least severely affected of the zinc-binding motif mutants, and dense nuclear aggregates were clearly associated with particulate forms. This reinforced the previous impression that the dense nuclear aggregates formed as a result of Gag3 accumulation and could represent a precondensation stage of particle morphogenesis.
FIG. 9. EM analysis reveals different Ty3 particle morphologies and localization controlled by NC function. BY4741 cells were transformed with the wt and Ty3 NC mutants, grown to log phase, and induced for Ty3 expression for 6 h at 24°C. Cultures were (more ...)
Cells expressing mutants ΔNTD and ΔNC were the only ones in which Ty3-RFP foci formed and were not consistently associated with P bodies. Ty3-MS2 foci were generally absent. Dense material and a few particles were observed in the nucleoplasm of these cells. A minority of cells showed small groups of cytoplasmic particles and individual particles lacking dense centers. However, a fraction of these cells also showed paracrystalline arrays. The diameter of 15 of the ΔNC particles was 40.7 ± 2.6 nm, indicating that they were also similar in diameter to wt VLPs. These also occurred in cells expressing the ΔNC(PR−) mutant. Taken together, these findings indicated that Ty3 can assemble into relatively stable, although not demonstrably separable, particles in the absence of the entire NC.