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1 Department of Basic Medical Sciences, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The class-II RFs, RF3 in bacteria and eRF3 in eukaryotes, are members of the GTPase family (Grentzmann et al. 1994; Mikuni et al. 1994; Stansfield et al. 1995). eRF3 is composed of two domains (Fig. 1A), C-terminal domain (referred to as CTD) containing the guanine nucleotide binding motif and N-terminal extension domain (referred to as NED). The CTD of eRF3 is highly conserved in its primary structure and is homologous to eukaryotic elongation factor eEF1
. On the other hand, the NED itself is not conservative in its primary structure; however, almost all the eRF3s so far reported possess NED regardless of being dispensable for cell growth (Ter-Avanesyan et al. 1993; Hoshino et al. 1998). The biological meaning of NED is poorly understood.
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Non-sense suppression assay is commonly used to estimate the translation termination efficiency (see references in Table 1). However, it is known that over-expression of an eRF-binding factor tends to cause artificial non-sense suppression by the deprivation of RFs rather than by altering the activity of RFs or ribosomes. Thus, it is difficult to elucidate molecular mechanisms behind non-sense suppression induced by over-expression of eRF-binding factors. The alternative approach to estimate the activity of RFs and the interplay of RFs with their binding factors is to use temperature sensitive (ts) mutants. In bacterial study, the selection for suppressors of ts lethal RF mutants gave rise to a variety of mutations in RF itself or in factors that interact with the RF, showing that the ts suppression phenotype is more likely to reflect the recovery of certain specific activity of its own (Matsumura et al. 1996; Sato et al. 2006). An important lesson from these genetic studies is that the mutational conditional activity, that is, the threshold for viability, is set mostly in such a narrow range that the influence of binding-factors on viability could be often detected sensitively. In view of the successful genetic study of isolating ts suppressors in bacteria, similar genetic approach in eukaryotes might provide us with an interesting insight into the functionality of eRFs and the interplay with their binding factors as well.
In this study, we prepared a series of yeast strains that express ts lethal eRF3 protein at three different levels, which represent distinct thresholds for viability. Using these strains we examined the effect of eRF3-binding factors on the viability of the test strains. Through this screen, we found that two eRF3-binding factors, Sla1 and Itt1, both of which are known to interact with the NED of eRF3, affect functionality of eRF3 positively or negatively. A regulatory role of NED through these factors is suggested.
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Results |
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The yeast strain YK21-02 (gst1-1) harbors a Tyr-to-Ser change at position 420 in eRF3, which confers ts lethality at 37 °C (Kikuchi et al. 1988). Since [PSI+] or [psi-] state of YK21-02 is not clearly described in the literature, YK21-02 was pretreated with guanidine hydrochloride and colonies formed were confirmed for a non-prion [psi-] state by fluorescent microscopy of the absence of eRF3 aggregates as previously described (Hara et al. 2003). The resulting [psi-] strain, HK100, was used throughout this study. When eRF1 was over-expressed in HK100 from a single copy plasmid vector (p416CYC1), the growth of the strain at 37 °C was significantly restored (Fig. 1B). This finding prompted us to use HK100s ts growth as a sensitive indicator of eRF3ts functionality that might alter, if any, upon factor binding.
Moreover, to detect up and down changes in eRF3ts functionality, we engineered by manipulating the expression level an intermediate condition of the eRF3ts availability that is susceptible to factor binding as to weaken or strengthen the ts lethality. The eRF3ts was placed under the relatively weak CYC1 promoter (Mumberg et al. 1995) and expressed from a single- or multi-copy plasmid in HK100. Transformants with empty, single- and multi-copy vectors are designated as HK100E, HK100S and HK100M, respectively. While HK100E failed to grow at 37 °C, HK100S grew weakly and HK100M grew quite well at 37 °C (Fig. 1C), suggesting that the eRF3ts protein possesses a residual weak activity at 37 °C and its gene dosage constitutes the rate limiting for cell growth. Under this condition, the eRF3ts protein abundance increased in proportion to the empty, single-copy and multi-copy plasmids as determined by Western blotting (shown later in Fig. 3). Therefore, we assumed that HK100E and HK100S might be useful to screen for factors that enhance eRF3ts functionality, while HK100S and HK100M for factors that reduce the functionality.
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We examined seven host factors that are known to interact with eRF3 (see Table 1). The first set of three, Itt1, Sla1, Pab1, binds to the NED, while the second set of four, Mtt1, Upf1 through Upf3, binds to the CTD. Plasmid-mediated over-expression and the pop-in/pop-out gene replacement for chromosomal knockouts, except for PAB1 (essential by itself) and UPF3 (its disruption in combination with eRF3ts was severely sick), were manipulated in HK100E, HK100S and HK100M. In over-expression experiments, the strong GPD promoter was used to over-synthesize eRF3 binding factors from a single-copy expression plasmid, except for Sla1 whose transformants from the GPD promoter were sick and unstable (data not shown). Instead, Sla1 was expressed from the ADH promoter that is 60 times weaker than the GPD (Mumberg et al. 1995). The resulting over-expression transformants and knockout constructs were established at permissive temperature 30 °C and examined for their growth at 37 °C (Fig. 2A,C,D; data summarized in Table. 2).
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). Itt1 over-expression eliminated the weak growth of HK100S and significantly weakened the growth of HK100M at 37 °C (Fig. 2A, left). No proper colonies formed in sla1 derivatives of HK100S and HK100M at 37 °C (Fig. 2A, right). All the other factors did not affect the growth phenotype (Fig. 2C,D). Itt1 over-expression and sla1 themselves are not toxic or sick at all in our wild-type eRF3 strains background at both 30 °C and 37 °C. In fact, transformation of these +Itt1 and sla1 variants of HK100E with a plasmid bearing wild-type eRF3 reversed the growth (Fig. 2E), showing that the primary cause of enhanced lethality was due to the reduced functionality of eRF3ts. This may not be inconsistent with the previous report that the sla1 disruption exhibits severe growth defect especially at high temperatures (Ayscough et al. 1999) by assuming a difference in the genetic background of strains used.
The next obvious question is whether or not Itt1 knockout (itt1) and Sla1 over-expression (+ Sla1) are able to restore ts growth as contrary to the growth defect enhanced by +Itt1 and sla1. Sla1 over-expression from ADH promoter showed no apparent change in growth phenotype of HK100E and HK100S (Fig. 2C). However, when Sla1 is expressed from weak CYC1 promoter (p416CYC) in the HK100 sla1 strain (HK102), transformants grew normally at 37 °C (Fig. 2B). Since the high expression of Sla1 from the strong GPD promoter was toxic to cells (data not shown), the moderate expression from the ADH promoter might be still partly toxic and counteract the positive influence. These findings suggest that Sla1 is a positive regulator of eRF3 function or availability. On the other hand, any phenotypic change was not detected with itt1 (see Fig. 2D). We speculate that the cellular abundance of Itt1 is limiting and/or there are redundant pathways for eRF3 destabilization, therefore its negative effect can be enhanced rather by over-expression.
Protein abundance of eRF3ts in +Itt1 and sla1 strains
One possibility of enhanced lethality of eRF3ts strain is an increased eRF3ts instability by +Itt1 and sla1. This gets some reality since mutant eRF3 proteins are often labile (Kong et al. 2004). HK100E, HK100S and HK100M strains carrying Itt1 over-espression plasmid or the sla1 allele in the chromosome were grown at 30 °C and total proteins were analyzed by Western blotting using anti-eRF3 antibody after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In control strains, the signal intensity of eRF3ts increased in proportion to the gene dosage (Fig. 3A, lanes 1–3, 7–9 and 13–15). This is clearly and quantitatively shown as values relative to those of Pgk1 that served as internal control (Fig. 3B, open boxes). When sla1 is nullified, the eRF3ts abundance went down significantly in HK100E, HK100S and HK100M (Fig. 3A, middle; Fig. 3B, middle). Similar instability of eRF3ts was observed upon over-expression of Itt1 in HK100E, HK100S and HK100M (Fig. 3A, left; Fig. 3B, left). In this regard, it is reported, and also confirmed by us, that over-expression of Itt1 causes non-sense suppression but does not affect cellular wild-type eRF3 abundance (Urakov et al. 2001). Therefore protein instability observed in our study might be amplified by ts lethal mutation in eRF3.
There is another evidence for the direct relationship between the eRF3ts protein abundance and cell viability. The optimal expression of Sla1 from plasmid p416CYC1 in strain HK102 (sla1) reversed the restricted growth as shown in Fig. 2B. When the same strain cultures were examined, the eRF3ts level strikingly increased (Fig. 3C), revealing that Sla1 functions to stabilize eRF3. Importantly, unlike Sla1 and Itt1, over-expression of eRF1 did not affect the abundance of eRF3ts (Fig. 3A, right; Fig. 3B, right). These findings are interpreted as indicating that stabilization and destabilization of eRF3ts by Sla1 and Itt1, respectively, are not achieved by the altered translation termination per se but are achieved by other cellular processes directly involving eRF3.
Evaluation of eRF3 stability upon removal of the NED
The results in this study so far shed light on the novel function of NED to regulate eRF3 protein stability through the interplay with, at least, Sla1 and Itt1. We then asked the intrinsic functionality of NED in eRF3 stabilization by removing the NED. The wild-type eRF3 (referred to as eRF3wt) and eRF3ts as well as their NED-less variants, eRF3wtNED and eRF3tsNED, were placed under the native SUP35 (i.e., eRF3) promoter in plasmid pRS313SUP35. These expression plasmids were introduced into the test strain HK116, in which chromosomal eRF3 gene was nullified by sup35::TRP1 and, instead, plasmid-encoded eRF3 was expressed from the GAL promoter for cell viability in the presence of galactose. In the absence of galactose, HK116 is lethal (Fig. 4A, strain 5). Transformants were incubated on galactose media until colonies were formed, and then shifted to glucose media. As shown in Fig. 4A, transformants with eRF3wt-, eRF3wtNED- and eRF3ts-bearing plasmids grew well in the absence of galactose at 30 °C. On the other hand, transformants with eRF3tsNED-bearing plasmid failed to grow under the same condition (Fig. 4A).
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NED is not viable (Fig. 4A), the amount of NED-less proteins was measured in the strains that carrying full-length eRF3 or eRF3ts. HK117 and HK118 cells were transformed with pRS313SUP35 plasmids carrying eRF3wtNED and eRF3tsNED, respectively, at 30 °C. Western blotting revealed that the cellular level of eRF3ts drastically decreased upon removal of NED, while eRF3wtNED abundance remained the same as eRF3wt (Fig. 4B). Under the same conditions, eRF3ts- and eRF3tsNED-mRNAs were unchanged (Fig. 4B).
To address the turnover rate of eRF3ts and eRF3tsNED proteins, protein levels upon addition of cycloheximide were monitored. eRF3ts and eRF3tsNED were expressed in HK100 from strong GPD promoter in p416 plasmid derivatives for sufficient detection of eRF3tsNED (note that a weak or moderate promoter is not sufficient to accumulate eRF3tsNED). Transformants were grown to log-phase at 30 °C and shifted to 37 °C, followed by addition of cycloheximide at the same time (time zero). Aliquots of cell cultures were harvested at 30, 60, 120, 240 and 540 min, and proteins were subjected to Western blotting using anti-eRF3 antibody. As shown in Fig. 4C, the estimated half-life of eRF3tsNED is about ninefold less than the full-length eRF3ts. These results indicate that NED itself possesses intrinsic potential to stabilize eRF3.
Itt1-induced eRF3ts instability monitored in the proteolysis deficient strains
To gain insight into the mechanism underlying the Itt1-induced eRF3ts instability, effects of proteolysis-deficient mutations were examined. Individual gene-knockouts in either one of the key components of major protein degradation pathways were introduced into chromosomes of the test strain HK100 bearing Itt1-over-expression plasmid. These include components for ubiquitin proteolytic pathway, DOA1, RPN10 and RPN13, as well as components for autophagy/vacuolar proteolytic pathway, ATG8, ATG12, ATG17, PEP4, PRB1 and PRC1. The former disruptants, doa1, rpn10 and rpn13, are known to accumulate polyubiquitinylated proteins (Ghislain et al. 1996; Verma et al. 2000; Saeki et al. 2002), while the disruptants of atg8, atg12 and atg17 are known to be defective in bulk protein degradation, and disruptants of vacuolar proteinase, pep4, prb1 and prc1, are also known to accumulate various proteins in the vacuole (Wang & Klionsky 2003).
HK100 derivative strains indicate weak but significant increase in colony formability at 37 °C with pep4, prb1 and prc1 (Supplementary Fig. S1), while other knockouts, except for doa1, did not affect the viability (data summarized in Supplementary Table S1). By unknown reason, the HK100 doa1 derivative exhibits severe growth defect at 37 °C at every eRF3ts expression level tested. These findings suggest that Itt1-induced eRF3(ts)-specific degradation may not be triggered by ubiquitination but can be processed, if not specifically through the autophagy (ATG) targeting pathways, by the vacuolar proteinase.
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Discussion |
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Interestingly, unlike Sla1 and Itt1, over-expression of eRF1 did not affect the cellular abundance of eRF3ts (Fig. 3B, right). This probably suggests that the alteration in eRF3 protein stability induced by manipulation of Itt1 and Sla1 genes involves a specific physiological pathway(s) that is independent of translation termination. One might argue that this speculation is contradictory to the previous observation that Itt1 is a novel factor to inhibit translation termination and its over-expression causes non-sense suppression (Urakov et al. 2001). However, we could say that non-sense suppression and anti-suppression do not always represent a direct functional change in the translation apparatus but in some cases are induced indirectly by an altered cellular abundance of translation factor(s) as shown in this study, and vice versa. In fact, some of the factors affecting non-sense suppression in previous reports did not affect significantly the eRF3ts-based cell viability assay developed here. For example, upf gene knockouts are known to cause non-sense mediated decay (Weng et al. 1996; Maderazo et al. 2000; Wang et al. 2001), but showed no influence in our assay (see Fig. 2 and Table 2). In this case, non-sense suppression by upf gene disruptions might be caused by the stabilization of reporter gene mRNA itself that contains premature non-sense mutations. Similarly, Upf1 over-expression leads to anti-suppression in NMD deficient yeast strains (Maderazo et al. 2000), yet no appreciable change was observed in our eRF3ts-based cell viability assay. Two other factors, Pab1 and Mtt1, behaved similarly, that is, anti-suppression or suppression induced by over-expression (Czaplinski et al. 2000; Cosson et al. 2002) while no effect on eRF3ts activity (see Fig. 2 and Table 2). These findings strongly suggest that the eRF3ts-based cell viability assay is sensitive and useful to detect primarily an altered protein state of eRF3.
Sla1 is a cytoskeletal protein that is required for cortical actin patch structure and its organization. Importantly, it is known that Sla1 functions as an adaptor to couple actin regulatory factors to the endocytic machinery (Ayscough et al. 1999; Warren et al. 2002). Moreover, there are several reports that indicate physiological relationships between eRF3 and cytoskeleton. Shortage of cellular eRF3 causes various defects in cytoskeleton (Tikhomirova & Inge-Vechtomov 1996). Mutations in RFs often show increased sensitivity to cytoskeletal inhibitory drugs (Valouev et al. 2002), and a number of other cases show functional linkages between cytoskeleton and translational events (Bailleul et al. 1999; Munshi et al. 2001; Liu et al. 2002; Valouev et al. 2004) as well as direct associations between Sla1 and translation-related factors (Gavin et al. 2006). Taking these results into consideration, we assume that the cytoskeletal network plays a crucial role to distribute cellular eRF3 to a proper cellular location where eRF3 is associated with other translation apparatus and thereby protected from proteolysis.
Itt1 is a member of the TRIAD family protein that possesses Ring-finger motifs (Urakov et al. 2001) and its function is not known. An interesting relevance between eRF3 and Ring-finger protein has been pointed out in human eRF3 (GSPT1). GSPT1 interacts with inhibitors of apoptosis proteins (IAPs) that includes Ring-finger motif with its specific site in NED, possibly regulating the target protein degradation and/or apoptosis (Hegde et al. 2003). In spite of the low sequence conservation among eRF3s from different species, it is likely that NED commonly serves as a site for binding to Ring-finger protein to couple eRF3 to a protein degradation pathway. Since the Ring-finger and the TRIAD family proteins are supposed to function as E3-ubiquitin ligase (Lorick et al. 1999), the ubiquitination dependent protein degradation pathway might be involved in Itt1-assisted eRF3 instability. However, a set of gene knockouts in ubiquitin-proteasome components, DOA1, RPN10 and RPN13, did not affect the Itt1-assisted eRF3 degradation, as well as the disruption of autophagy components, ATG8, ATG12 and ATG17, which is responsible for bulk protein degradation pathway. Instead, the Itt1 effect in eRF3 instability was significantly prevented by gene knockouts in vacuolar proteinases, PEP4, PRB1 and PRC1 (Supplementary Fig. S1), suggesting a novel aspect of Ring finger protein family in protein degradation. Though the exact pathway of enhanced protein degradation is still obscure, it is certain that Itt1 expression actually alter the stability of eRF3ts.
The remaining question is whether or not NED-catalyzed eRF3 (in)stability is significant not only with the eRF3ts allele but also with wild-type eRF3 in general. Although this remains to be investigated, we believe that the thermo-labile eRF3ts proteins facilitated the detection of the eRF3 decay pathway occurring in the wild-type strains. In fact, there are some cases to point out the functional significance of NED of eRF3wt. The wild-type S. pombe eRF3 can functionally substitute for the S. cerevisiae eRF3, while NED-less S. pombe eRF3 became severely labile and failed to function properly in S. cerevisiae (Kong et al. 2004). This is comparable to the result here that the NED-less eRF3ts protein became severely labile and no longer functioned (Fig. 4). The eRF3ts allele is a Tyr-to-Ser substitution in the GTP binding consensus domain (Fig. 1A) (Kikuchi et al. 1988). Therefore, it is conceivable that the mutation affects GTP turnover so severely that the mutant protein could not be recruited in the translation machinery efficiently. Given this scenario, we might speculate that the (in)stability control by NED-binding factors could be a general property for eRF3 protein and be more prominent in certain harsh conditions in which protein synthesis is restrictive. Otherwise, it is likely that the protein instability of eRF3ts might represent one of the discrete functional modes of cellular eRF3 proteins altered by binding factors, in which protein degrading machinery can access specifically to abnormally labile molecules that are apparently enhanced with eRF3ts mutation and seemingly cryptic in wild-type eRF3. Further studies are necessary to uncover these intriguing possibilities.
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Experimental procedures |
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The yeast strains used in this study are listed in Table 3. To confirm the [psi-] state of yeast strain, we passaged YK21-02 cells on YPD medium containing 3 mM guanidine hydrochloride (GuHCl) and subsequently passaged cells on GuHCl-free YPD media. Yeast cultures were grown using standard conditions in YPD liquid medium (2% w/v Bacto-peptone, 1% w/v yeast extract and 2% w/v glucose). Yeast transformants were grown in synthetic complete (SC) media containing glucose or galactose as a carbon source, supplemented with the required amino acids and cofactors. Knockout alleles of eRF3-binding factors were newly constructed in this study by substitution of the relevant disruptions (KanMX markers) from Saccharomyces Genome Deletion Project <http://sequence-www.stanford.edu/group/yeast_deletion_project/>. The integrative construct carrying the relevant ORF::KanMX cassettes were amplified by polymerase chain reaction (PCR) with sets of primers and transformed into HK100 strain. Disruptants were selected on YPD media containing 0.2 mg/mL G418 sulfate and the ORF::KanMX replacement into the chromosome of the parental HK100 strain in place of endogenous genes was confirmed by genomic PCR.
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For the indicated factors used for growth assay, the corresponding genes were amplified by PCR from yeast genomic DNA using sets of primers. The amplified fragments were subcloned into the pT7 Vector by blunt end cloning, then transferred into appropriate plasmid, p414/424 or p416/426, suitable for an amino acid marker and a promoter strength (CYC: weak, ADH: intermediate, GPD: strong and SUP35 native promoter).
Primers
Primer sequences are available upon request. In brief, sets of primers for gene disruption were designed by sequences 50–300 base pairs upstream or downstream of each gene. All primers for gene expression were designed to have EcoRI, BamHI or HindIII site for 5' primers and SalI or XhoI site for 3' primers, respectively.
Western blots
Total proteins were prepared from yeast cells grown to mid-log phase in appropriate media at 30 °C. Protein concentration was determined by means of a Bio-Rad Protein Assay (Bio-Rad Laboratories). Proteins were separated by SDS-PAGE using 7.5% polyacrylamide gels and transferred to ImmobilonTM-P Transfer Membrane (Millipore). For eRF3 detection, the blots were incubated with rabbit anti-eRF3 antibody (Hara et al. 2003), followed by incubation with peroxidase-conjugated anti-rabbit-IgG secondary antibody (GE Healthcare Bio-Sciences) and detected with ECL Western blotting detection/analysis system (GE Healthcare Bio-Sciences) according to the manufacturer's instructions. For Pgk1 detection, the blots were incubated with mouse anti-Pgk1 antibody (Invitrogen). Followed by incubation with peroxidase-conjugated appropriate secondary antibody (GE Healthcare Bio-Sciences) and detected similarly.
Northern blots
Total RNAs were prepared from cells grown to mid-log phase in appropriate media, extracted with phenol/chloroform then precipitated with ethanol. The total RNAs were resolved by 1.2% agarose-gel electrophoresis in the presence of formaldehyde and blotted on to Hybond-N +membrane (GE Healthcare Bio-Sciences). For mRNA visualization, the blots were incubated with DNA probe for eRF3 C-terminal region that was modified with peroxidase and detected by ECL RNA detection system.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: itopi005{at}ims.u-tokyo.ac.jp
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References |
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Received: 24 January 2007
Accepted: 13 February 2007
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