Dissecting toxin immunity in -infected killer uncovers an intrinsic strategy of self-protection

Frank Breinig, Tanja Sendzik, Katrin Eisfeld*, and Manfred J. Schmitt†

Angewandte Molekularbiologie, Universita¨t des Saarlandes, D-66041 Saarbru¨cken, Germany

Edited by Randy Schekman, University of California, Berkeley, CA, and approved January 6, 2006 (received for review November 21, 2005) Toxin-secreting ‘‘killer’’ were initially identified >40 years toxin’s ␤ subunit that enables the toxin to reach the secretory ago in strains infected with a double- pathway from an early endosomal compartment (14). Once within stranded RNA ‘‘killer’’ virus. Despite extensive research conducted the ER, the ␣͞␤ heterodimer dislocates into the cytosol via Sec61c, on yeast killer toxins, the mechanism of protecting immunity by the major ER transport channel, subsequently transmitting its lethal which toxin-producing cells evade the lethal activities of these signal into the nucleus. proteins has remained elusive. Here, we identify the mechanism Extensive investigation of the killer phenomenon in yeast has leading to protecting immunity in a killer yeast secreting a viral ␣͞␤ resulted in substantial progress in elucidating many of the intricacies protein toxin (K28) that enters susceptible cells by -medi- of this phenomenon and, additionally, providing valuable insight ated endocytosis and, after retrograde transport into the cytosol, into a number of fundamental aspects of eukaryotic biology and blocks DNA synthesis, resulting in both cell-cycle arrest and virus–host cell interactions (3, 6, 15). Furthermore, the detailed caspase-mediated . We demonstrate that toxin immunity analysis of the toxins’ mode of action also revealed a number of is effected within the cytosol of a toxin-secreting yeast and occurs potential targets against which antifungal agents could potentially via the formation of complexes between reinternalized toxin and be directed. Despite all this, the mechanism by which toxin- unprocessed precursor moieties that are subsequently ubiquiti- producing cells evade the lethal activities of these proteins has nated and proteasomally degraded, eliminating the active form of remained elusive, especially since the recently proposed model for the toxin. Interference with cellular ubiquitin homeostasis, either K1 immunity (16) has, meanwhile, been repeatedly shown to be of through overexpression of mutated ubiquitin (Ub-RR48͞63)orby little significance in vivo (11, 17, 18). Until the present study, no blocking deubiquitination, prevents ubiquitination of toxin and attempt has been made to explain how K28 toxin-producing cells results in an impaired immunity and the expression of a suicidal resist the activity of this protein. In this investigation, we show that phenotype. The results presented here reveal the uniquely elegant K28-producing cells, just like toxin-susceptible target cells, take up and efficient strategy that killer cells have developed to circumvent toxin and transport it to the cytosol. We demonstrate that immunity the lethal effects of the toxin they produce. to K28 does not rely on inhibition of extracellular toxin uptake and present data revealing that both pptox and reinternalized mature ␣͞␤ protein toxin ͉ preprotoxin ͉ ubiquitination toxin are present in the cytosol of K28-producing cells. Replace- ment of the N-terminal K28 leader peptide by the cotranslationally he killer phenomenon in yeast, originally discovered in certain active K1 pptox signal peptide indicated that the mechanism of K28 killer strains of Saccharomyces cerevisiae in 1963, is associated pptox import into the ER is critical in conferring toxin immunity. T Partial immunity to K28 could be achieved by cytosolic expression with the of a protein toxin that kills sensitive target cells ␣ in a receptor-mediated fashion without direct cell-to-cell contact (1, of only the mature subunit of the toxin, whereas complete protection required a nonspecific sequence extension to the C 2). In baker’s yeast, the killer phenotype depends on the presence ␣ of cytoplasmic dsRNA killer encoding the unprocessed terminus of the subunit. Finally, we reveal the formation of a toxin precursor protein (3). Until now, three killer toxins have been complex between pptox and the reinternalized, mature toxin in the cytosol of K28-producing cells and demonstrate that, in vivo, identified, of which K1 and K28 have been studied most extensively. ͞ Each killer toxin is translated as a preprotoxin (pptox) precursor ubiquitination and proteasomal degradation of this pptox K28 that is imported into the yeast secretory pathway where it is complex is essential for K28-producing cells to achieve complete processed to the mature and cytotoxic ␣͞␤ heterodimer, which is protection and immunity from the toxin. then released from the cell and secreted into the medium (4–6). Results and Discussion Although the two ‘‘prototypes’’ of yeast killer toxins K1 and K28 exert their lethal effect in a receptor-mediated fashion, the mech- Immune Killer Cells Partially Reinternalize Secreted Toxin. In killer anism of toxin-induced lethality is significantly different. K1 dis- yeast, functional immunity is central and essential for cell survival, rupts cytoplasmic membrane function, whereas K28 enters its because the toxins exclusively target and inhibit eukaryotic cell target cell by receptor-mediated endocytosis and blocks DNA functions. This situation is in major contrast to protein toxins synthesis, leading to both G1͞S cell-cycle arrest and caspase- produced by bacteria (such as cholera toxin and Shiga toxins) which mediated apoptosis (7, 8). Both toxins initially bind to a primary selectively kill eukaryots, thus making immunity and self-defense in receptor within the yeast cell wall and are then translocated to the a prokaryotic host dispensable. plasma membrane, where they interact with a secondary toxin The K28 toxin, in common with cholera toxin, Pseudomonas receptor (9, 10). The glycosylphosphatidylinositol-anchored cell-

surface protein Kre1p has recently been identified as the plasma Conflict of interest statement: No conflicts declared. membrane receptor for K1 toxin, whereas the membrane receptor This paper was submitted directly (Track II) to the PNAS office. for K28 remains unknown (11). After having bound to the yeast cell Abbreviations: ER, ; pptox, preprotoxin; ptox, protoxin; ⌬tox, pptox surface, K1 perturbs plasma membrane function by forming cation- variant lacking its N-terminal prepro sequence; Ub, ubiquitin. selective ion channels (12, 13), whereas K28, in contrast, enters the *Present address: Institut fu¨r Biotechnologie und Wirkstoff-Forschung, D-67663 Kaiser- cell and is trafficked through the secretory pathway to translocate slautern, Germany. into the cytosol and reach its final target in the nucleus. Retrograde †To whom correspondence should be addressed at: Angewandte Molekularbiologie, FR toxin transport from and through the and the 8.3, Geba¨ude A 1.5, Universita¨t des Saarlandes, Postfach 151150, D-66041 Saarbru¨cken, endoplasmic reticulum (ER) critically depends on the presence of Germany. E-mail: [email protected]. a short amino acid sequence (HDEL) at the C terminus of the © 2006 by The National Academy of Sciences of the USA

3810–3815 ͉ PNAS ͉ March 7, 2006 ͉ vol. 103 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510070103 Downloaded by guest on September 28, 2021 Fig. 1. Posttranslational pptox import into the ER and rein- ternalization of the mature ␣͞␤ toxin. (A) SDS͞PAGE and Western blot analysis of the intracellular membrane fraction (M) and the cytosol (C) of the K28 superkiller mutant (ski2–2) S. cerevisiae MS300b and its heat-cured nonkiller derivative probed with a polyclonal anti-␤-subunit antibody. Positions of the unprocessed pptox in the cytosol, the N-glycosylated ptox in the ER lumen, and the ␣͞␤ heterodimeric K28 toxin in toxin-treated (ϩK28) and untreated (ϪK28) cells are indi- cated. Organellar marker proteins in the same subcellular fractions of the K28 killer strain MS300b were probed with anti-Kar2p (ER lumenal marker), anti-Sec61p (ER membrane marker), and anti-Pgk1p (cytosolic marker). (B) Immunoblot of secreted K28 toxin in cell-free culture supernatants of the temperature-sensitive yeast mutants sec71, sec72, sec62, sec63, and sec61 and their isogenic SEC wild-type equivalent after cultivation at the semirestrictive temperature of 30°C. Positions of the ␣͞␤ heterodimeric toxin and its tetrameric derivative (␣͞␤)2 (which forms spontaneously under condi- tions of a nonreducing SDS͞PAGE) are indicated. (C) Schematic outline of K28 pptox derivatives containing either the natural N-terminal secretion signal (prepro sequence) of K28 pptox or the corresponding prepro or pre sequence of K1 pptox (shown in a black box) under transcriptional control of the galactose- inducible GAL1 promoter and their phenotypic effect on toxin immunity after in vivo expression in the K28-sensitive strain S. cerevisiae SEY6210. Immunity against exogenously applied K28 toxin was determined on MBA plates (pH 4.7) under repressing or inducing culture conditions in the presence of glucose or galactose as carbon source. (D) Model of posttrans- lational pptox import into the ER lumen. In the cytosol of a toxin-producing killer yeast, unprocessed pptox encoded by the killer viral M(ϩ)ssRNA transcript enters the secretory path- way through the major ER import channel (Sec61 complex) aided by cytosolic chaperones of the Ssa family (not indicated). Consequently, pptox import into the ER is prevented or at least significantly impaired in the indicated conditional yeast mutants sec61, sec62, sec63, sec71, and sec72. SV, secretory vesicles.

exotoxin A, and other family members of microbial ␣͞␤ toxins, uses mutant defective in early steps of endocytosis (data not shown). The receptor-mediated endocytosis and retrograde transport to reach presence of significant amounts of the ␣͞␤ heterodimeric toxin in its intracellular target compartment (14). In principal, therefore, the cytosol of K28-producing cells supports the argument that the toxin immunity could be conferred either by preventing reinter- dimeric conformation of the mature toxin is rather stable, a finding nalization of secreted, active toxin, or by inactivating reinternalized that is in agreement with recent studies on human superoxide toxin somewhere within the secretory pathway or further down- dismutase that similarly appears to have the ability to maintain stream in the yeast cell cytosol. By analyzing the intracellular intrasubunit disulfide bonds in the reducing environment of the membrane fraction and the cytosol of a K28-secreting superkiller cytosol (20). This finding also confirms that K28 toxin’s retrotrans- (ski2–2) mutant, we show that both the pptox precursor protein and location from the ER to the cytosol occurs in the ␣͞␤ heterodimeric the mature and fully processed ␣͞␤ heterodimer are present in the conformation (14) and is consistent with a recent report that, cytosol, whereas an additional 48-kDa toxin signal is detectable in likewise, showed that protein unfolding is not a prerequisite for ER the intracellular membrane fraction, most likely representing the dislocation (21). The conclusion from these results is that reinter- N-glycosylated protoxin (ptox) resulting from signal peptidase nalization of at least a fraction of the active ␣͞␤ toxin occurs in MICROBIOLOGY cleavage and coreglycosylation in the ER lumen (Fig. 1A). The K28-producing cells and that immunity relies on inactivation of small amount of ptox present in the yeast cell cytosol indicates some reinternalized toxin rather than its exclusion from the cell. ptox leakage from the secretory pathway during cell lysis (Fig. 1A); however, this minor amount does not account for the observed Posttranslational pptox Import into the ER Is Critical for Toxin pptox enrichment in the cytosol. As expected, no such toxin-specific Immunity. Our finding of significant amounts of pptox within the signal was detectable in intracellular membranes of the correspond- yeast cell cytosol implies that this protein is imported posttransla- ing nonkiller derivative obtained after heat-curing the ski2–2 mu- tionally into the secretory pathway (representing the predominant tant from its toxin-encoding M-dsRNA killer virus (19) (Fig. 1A). mechanism of protein import into the ER in yeast) rather than Interestingly, when the same killer cells were treated with exoge- cotranslationally, as it preferentially occurs in mammalian cells nous toxin, the amount of mature ␣͞␤ toxin present in the cytosol (22). Detailed analysis of the physicochemical properties of the K28 significantly increased, indicating that K28 killer cells are capable of signal peptide as well as calculations of the hydrophobic core values taking up their own toxin and transporting it retrograde to the both supported our prediction that pptox import into the ER occurs cytosol, just like toxin-susceptible nonkiller cells (ϩK28 in Fig. 1A). posttranslationally (data not shown). In support of the in silico data, This observation also implies that K28 immunity does not rely on we found a dramatic reduction of K28 secretion in conditional inhibition of extracellular toxin uptake; and, vice versa, toxin mutants impaired in essential cellular components of posttransla- reinternalization was completely blocked in a K28-secreting end3ts tional protein import into the secretory pathway. As illustrated in

Breinig et al. PNAS ͉ March 7, 2006 ͉ vol. 103 ͉ no. 10 ͉ 3811 Downloaded by guest on September 28, 2021 Fig. 1B, K28 toxin secretion was almost completely blocked in yeast sec71 and sec72 mutants and significantly decreased in sec62, sec63, and sec61 mutants when cultivated at the semirestrictive tempera- ture. The most pronounced decrease in toxin secretion was seen in sec71, sec72, and sec62 mutants, whose gene products are believed to form a cytosolic receptor complex involved in the signal- recognition particle-independent posttranslational protein import into the ER lumen (23, 24). In addition, toxin secretion was markedly decreased in mutants defective in cytosolic Hsp70 chap- erones (such as Ssa1p and Ssa2p) that are known to be required for posttranslational translocation into the ER (data not shown). Interestingly, substitution of either the K28 signal peptide or the entire K28 prepro region with the corresponding sequence(s) of the significantly more hydrophobic K1 signal peptide (thus favoring cotranslational translocation into the secretory pathway) led to a dramatic increase in K28 susceptibility of toxin-producing cells harboring the chimeric K1͞K28 construct (Fig. 1C), demonstrating that a certain level of pptox in the cytosol of a killer cell is necessary for the conferrence of immunity and that this level is maintained in vivo by posttranslational pptox translocation into the ER (Fig. 1D). This finding is in agreement with previous findings showing that functional immunity occurs only if pptox expression exceeded a certain critical level (25, 26). Our results provide striking evidence that the mechanism of pptox import into the secretion pathway and its impact on the steady-state concentration of pptox in the cytosol is of critical importance in the ability of cells to exhibit immunity from K28 toxin.

The ␣ Subunit of K28 Is Essential for Immunity. The existence of pptox and reinternalized toxin within the cytosol of K28-immune cells and the finding that cellular uptake of external toxin is unaffected by the immune status of the cell suggested to us that the key element in ␣ toxin immunity involves an interaction between pptox and reinter- Fig. 2. The subunit of K28 is essential but not sufficient to confer protective ␣͞␤ immunity. Schematic drawing and effect on toxin immunity of K28 wild-type nalized mature toxin in the cytosol of toxin-producing cells. To pptox and various truncated variants thereof lacking the N-terminal prepro test this hypothesis, an N-terminally truncated K28 pptox derivative sequence after in vivo expression in the K28-susceptible strain S. cerevisiae lacking its natural prepro sequence and, thus, incapable of entry SEY6210. In the K1͞K28 chimeric constructs, the corresponding subunits from into the secretory pathway, was expressed in a K28-susceptible K1 pptox are indicated by a black box. In each case, pptox expression was strain under transcriptional control of the GAL1 promoter. As driven from the GAL1 promoter, and K28 immunity was determined in a illustrated in Fig. 2, the former K28-susceptible cells were immune well-plate assay on MBA (pH 4.7) with galactose as carbon source. In the pptox under induced conditions in the presence of galactose, whereas the variants ␣͞␤K-0, ␣K-0͞␤K-0 and pptoxK-0, the single lysine residue in ␤ or all same cells remained toxin-sensitive when pptox transcription was internal lysyl residues in either both subunits or in the entire preprotoxin had ␣͞␤ repressed (data not shown). To define the pptox domain that is been converted to arginine to reduce or prevent lysine-mediated ubiq- uitination in vivo (see also Fig. 3G). The particular toxin construct producing required for protective immunity, we independently introduced a the exemplarily shown halo on MBA is indicated in the figure. Note that number of constructs expressing different pptox domains into toxin-treated cells of K28-␣-expressing yeast show a small but cell-free zone of susceptible host cells and assessed the resultant transformants for growth inhibition around the well, whereas pptoxK-0-expressing cells show a toxin immunity. As shown in Fig. 2, expression of neither the ␤ dark blue colony staining around the well, indicating that the cells are being subunit nor the ␥ sequence that bridges ␣ and ␤ had any impact on killed by the toxin. toxin susceptibility, indicating that both subunits are not involved in conferrence of immunity. This finding bolsters our previous spec- ulation that ␤ functions as intracellular transport ‘‘vehicle’’ solely, of K1. Although both ␥ sequences share no sequence homology, ensuring proper uptake and efficient transport of the cytotoxic ␣ K28-susceptible transformants expressing ␥ from K1 were fully toxin to its final site of action, whereas ␤ has no relevance for the immune to K28 just as cells expressing ␣␥ from K28 but containing expression of protecting immunity. In contrast, sole expression of ␤ from K1 pptox (Fig. 2), substantiating the finding that expression ␣ rendered cells considerably less susceptible to toxin (Fig. 2), of C-terminal extended ␣ is both necessary and sufficient to confer demonstrating that the presence of ␣ is necessary, but not sufficient, protective immunity to K28. A similar phenomenon has been to confer complete immunity in vivo. Because, in the native pptox, described for the expression of immunity from K1, which likewise the ␣ subunit is C-terminally linked to the ␥ sequence, we investi- requires expression of a C-terminally extended ␣ subunit. For gated whether extending the C terminus of ␣ would increase the expression of K1 immunity, however, this C-terminal extension of level of K28 immunity. An additional toxin derivative in which the ␣ must, at a minimum, constitute the N-terminal 31 residues of the intervening ␥ sequence was removed, resulting in a direct linkage K1 ␥ subunit (27), whereas the C-terminal extension necessary for between ␣ and ␤, conferred complete immunity to K28-susceptible immunity from K28 appears to be essentially sequence-nonspecific cells (Fig. 2), confirming that a C-terminal extension of ␣ is needed and is unquestionably independent of ␥. to give full protection. Interestingly, because this construct con- tained a sequence that never occurs in nature, i.e., the ␣ subunit Cytosolic pptox Complexes with Reinternalized K28 Toxin. We had directly linked to ␤, the precise sequence of the C-terminal exten- shown that expression of unprocessed K28 pptox is sufficient to sion of ␣ is apparently irrelevant with respect to the conferrence of confer immunity by introducing a K28 cDNA into a susceptible immunity. To further confirm this interesting observation, we ⌬kex2 null mutant, incapable of pptox processing, and thereby replaced the ␥ sequence of K28 with the corresponding sequence generating immune nonkiller cells (28, 29). This finding is entirely

3812 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510070103 Breinig et al. Downloaded by guest on September 28, 2021 Fig. 3. Functional immunity requires ubiq- uitination and proteosomal degradation of the pptox͞K28 complex. (A) Schematic draw- ing of a His-tagged K28 pptox derivative lacking its N-terminal prepro sequence [⌬tox(His)6] and experimental setup to copu- rify a ⌬tox(His)6͞toxin complex in vivo. Yeast transformants expressing the His-tagged K28 toxin precursor ⌬tox(His)6 were sphero- plasted and treated with either native toxin (K28) or heat-inactivated toxin (K28HI), the latter being completely blocked in cell entry. Cells were lysed, cell debris and membranes were removed, and the resultant superna- tant applied onto a TALON column to purify the His-tagged K28-derivative. (B) Demon- stration of a ⌬tox(His)6͞␣͞␤ complex purified from the cytosol of toxin-immune cells. After purification, eluted proteins from A were fractionated by SDS͞PAGE and probed with polyclonal anti-␤ subunit antibody. Positions of the His-tagged protoxin [⌬tox(His)6] and the heterodimeric ␣͞␤ toxin are indicated. (C) Evidence that the ⌬tox(His)6͞␣͞␤ complex is ubiquitinated and proteasomally de- graded. Eluted protein samples from A were fractionated by SDS͞PAGE and detected by using polyclonal anti-Ub antibody. Positions of the ubiquitinated toxin precursor [⌬tox- (Ub)x] and the breakdown products of the ubiquitinated ⌬tox͞␣͞␤(Ub)x complex are in- dicated. (D) Western blot analysis of secreted K28 toxin and (E) invertase Suc2p in cell-free culture supernatants. (F) Impaired toxin im- munity in conditional proteasome mutants pre1–1 and pre2–2 and a ⌬doa4 knockout compared with wild-type. As negative control for invertase secretion, the ⌬suc2 mutant S. cerevisiae SEY6210 was used. (G) Relative amount of secreted K28 wild-type toxin (␣͞␤) and its two lysine-free variants ␣͞␤K-0 and ␣K-0͞␤K-0 in S. cerevisiae SEY6210 and decreased ␣͞␤ toxin secretion in a yeast ⌬doa4 mutant defective in protein deubiquitination compared with the isogenic DOA4 wild type. Positions of the ␣͞␤ heterodimeric toxin and its tetrameric 2ϩ deriative (␣͞␤)2 are indicated. (H) Lack of functional immunity and decrease in toxin secretion in K28 pptox-expressing wild-type cells before and after Cu -induced overexpression of mutated Ub (Ub-RR48/63) incapable of forming polyubiquitin chains.

consistent with, and supportive of, our hypothesis that the forma- sufficient processed toxin to kill susceptible cells. Our experiments tion of a complex between pptox and reinternalized K28 within the indicate that this is accomplished by ubiquitination and proteaso- cytosol of toxin-producing cells is the key step in immunity, because mal degradation of, primarily, the K28 component of the pptox͞ the formation of such a complex would occur irrespective of the K28 complex and by simultaneously ensuring that sufficient free cells’ ability to process pptox for secretion. We succeeded in pptox is available for (i) complex formation with reinternalized ␣͞␤ isolating the pptox͞K28 complex by copurifying a His-tagged pptox toxin and (ii) import into the ER and subsequent toxin secretion. derivative [⌬tox(His)6] lacking the N-terminal prepro sequence When K28-immune cells were treated with heat-inactivated toxin, (Fig. 3A) but still capable of conferring immunity (see Fig. 3), with several high-molecular-weight protein signals comprising ubiquiti- the mature ␣͞␤ heterodimer of K28. As shown in Fig. 3B, ⌬tox- nated forms of ⌬tox(Ub)x were visible (Fig. 3C). Furthermore, the (His)6 and heterodimeric toxin were copurified (albeit not in a cytosol of immune cells treated with native toxin additionally stoichiometric ratio; see below), providing evidence for the in vivo contained low-molecular-weight toxin signals that most likely rep- formation of a complex between K28 pptox and reinternalized resent proteasomal breakdown products of the heterodimeric toxin toxin. That this complex is formed between ⌬tox and reinternalized within the ⌬tox͞K28 complex (Fig. 3C). It appears that ubiquiti- toxin was also evidenced by its absence when heat-inactivated toxin nation and proteasomal degradation are somehow more effective to MICROBIOLOGY (K28HI in Fig. 3B) was used in lieu of native toxin. In contrast to the toxin component within the ⌬tox͞K28 complex, because ubi- native toxin, heat-inactivated K28HI is incapable of entering and quitinated degradation products in the low-molecular-weight range penetrating a sensitive target cell, because initial toxin binding to were hardly present in control cells treated with heat-inactivated both the K28 cell wall and its membrane receptor on the yeast cell ␣͞␤ toxin incapable of cell entry (Fig. 3C). In direct support, surface is completely blocked (J. Spindler and M.J.S., unpublished cytosolic ⌬tox complexed with reinternalized ␣͞␤ toxin was affinity- data). The in vivo formation of a pptox͞K28 complex presumably purified in a nonstoichiometric ratio (Fig. 3B), suggesting that an enables highly effective interception of reinternalized ␣͞␤ in toxin- excess in free cytosolic pptox in vivo exists, which similarly ensures producing cells immediately after this molecule has been retrograde pptox import into the ER and subsequent secretion of mature ␣͞␤ transported to the cytosol and before the release of the biologically toxin. Thus, it can be assumed that, once the ubiquitinated ␣͞␤ active ␣ subunit from the heterodimer. component of the pptox͞K28 complex has been proteasomally degraded, sufficient nonubiquitinated pptox is still available to Ubiquitination and Proteasomal Degradation of the Cytosolic pptox͞ either complex with another K28 molecule or enter the yeast K28 Complex Is an Essential Prerequisite for Functional Immunity. The secretory pathway to be processed into mature toxin; and, vice formation of cytosolic pptox͞K28 complexes raises the question of versa, it can be presumed that toxin secretion should be negatively how toxin-producing cells maintain adequate concentrations of free affected when proteasomal degradation of the complex is disturbed, pptox in the cytosol to ensure ongoing immunity while still secreting because, in this case, pptox primarily has to complex internalized

Breinig et al. PNAS ͉ March 7, 2006 ͉ vol. 103 ͉ no. 10 ͉ 3813 Downloaded by guest on September 28, 2021 Fig. 4. Mechanistic model of toxin immunity in a K28- secreting killer yeast. In the cytosol of a toxin-producing killer cell, the unprocessed preprotoxin encoded by the M-dsRNA killer virus complexes with mature ␣͞␤ toxin that has been reinternalized and retrogradely transported through the se- cretory pathway (1, 2). Within the subsequently generated complex of pptox and mature ␣͞␤ toxin (3), the ␤-subunit is (poly)ubiquitinated (Ubx) and degraded by the proteasome (4). Because of an excess in the noncomplexed and nonubiq- uitinated pptox in the cytosol, the toxin precursor can also be imported into the ER, processed, and secreted as active toxin (5).

toxin instead of being translocated into the ER. Such a scenario mal degradation of cytosolic pptox͞K28 complexes is not simply a should bias the balance between pptox needed to complex toxin and process to eliminate waste byproducts of K28-toxin biosynthesis but, pptox destined for ER import to pptox͞␣͞␤ complex formation. rather, is an essential and integral component in maintaining Consistent with this hypothesis is our finding that toxin secretion is immunity to toxin. However, in comparison with the immunity- significantly perturbed and immunity severely impaired in condi- compromised yeast mutants ⌬doa4 and͞or pre1͞2, the observed tional proteasomal yeast mutants (pre1–1 and pre2–2), resulting in decrease in toxin secretion was significantly more pronounced in the expression of a suicidal phenotype against exogenously applied wild-type yeast expressing either mutant Ub (Ub-RR48/63)oraly- toxin. At the restrictive temperature, these mutants are severely sine-free K28 derivative (pptoxK-0), indicating that the levels impaired in the chymotrypsin-like activity of the 20S proteolytic of toxin secretion and toxin immunity do not strongly correlate core particle of the proteasome (30), and cells of both pre mutants in vivo. show significantly decreased levels of toxin secretion and exhibit Based on the data presented here, we can now provide a increased susceptibility to toxin, although protein secretion in mechanistic model of the events resulting in immunity of K28- general is not negatively affected, as could be shown by SDS͞PAGE secreting cells from the action of this toxin (Fig. 4). We present and Western blot analysis of yeast invertase (Suc2p) secretion in evidence that immunity from K28 toxin, one of the three best pre1͞2 mutants compared with isogenic PRE1͞2 wild-type cells characterized toxins produced by virus-infected killer strains of S. (Fig. 3 D–F). This finding indicates that both toxin secretion and cerevisiae, occurs via a specific interaction between reinternalized immunity are severely impaired when cells are blocked (or at least toxin and its precursor protein within the cytosol of toxin-producing impaired) in proteasomal degradation of the cytosolic pptox͞K28 cells. This interaction results in the generation of a pptox͞K28 immunity complex. Interestingly, an entirely analogous phenotype complex that is a target for ubiquitination and subsequent protea- could be seen in cells expressing a mutant K28 variant in which somal degradation. This elegant, efficient, and entirely self- either the single lysine residue in ␤ or all internal lysyl residues in contained mechanism enables K28-producing cells to inactivate both subunits ␣ and ␤ had been destroyed and converted to arginine toxin before the cytotoxic ␣ toxin reaches its ultimate intracellular to prevent (or at least reduce) lysine-mediated ubiquitination and target. The mechanism of posttranslational pptox import into the proteasomal degradation in vivo. In case of ␣K-0͞␤K-0, secretion of yeast secretory pathway ensures that free cytosolic pptox is in the mutant toxin was significantly decreased compared with wild- significant excess over reinternalized ␣͞␤ toxin, and, thus, the type ␣͞␤ toxin (Fig. 3G), and functional immunity against exoge- formation of a pptox͞K28 immunity complex in the cytosol does not nous ␣͞␤ was negatively affected in cells expressing a K28 pptox negatively impact toxin production. variant completely avoid of internal lysyl residues (see Fig. 2). In sum, from these findings, it can be deduced that prevention of Materials and Methods ubiquitination of the reinternalized ␣͞␤ toxin within the cytosolic Yeast Strains and Culture Media. For the in vivo expression of the pptox͞K28 complex severely affects both toxin secretion and func- K1͞K28 pptox constructs, S. cerevisiae strain SEY6210 (MAT␣ tional K28 immunity. In a compelling parallel experiment, loss of leu2–3 his3-⌬200 lys2–801 trp1-⌬901 suc2-⌬9) was used. Cells were toxin immunity could also be accomplished by interfering with in grown in yeast extract͞peptone͞dextrose medium at 30°C. Trans- vivo ubiquitin (Ub) homeostasis. This result was achieved in two formation was carried out by the lithium acetate method according ways: first, by copper-induced overexpression of a mutated Ub to Ito et al. (31), and transformants were selected on synthetic (Ub-RR48/63) that can no longer form polyubiquitin chains and, complete medium lacking uracil. Yeast proteasomal mutants second, by deletion of a gene, DOA4, which encodes the deubiq- pre1–1 and pre2–2 were kindly provided by Dieter Wolf (University uitinating enzyme required for regenerating Ub from proteasome- of Stuttgart) (32); wild-type strain BY4742 (MAT␣ his3⌬1 leu2⌬0 bound ubiquitinated intermediates. Both scenarios resulted in the lys2⌬0 ura3⌬0) and all deletant strains were from the Saccharomy- expected loss in toxin immunity and decrease in toxin secretion ces Genome Deletion Consortium and were obtained from Re- (Fig. 3 G and H), demonstrating that ubiquitination and proteaso- search Genetics. The yeast ⌬doa4 null mutant MHY623 (MAT␣

3814 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0510070103 Breinig et al. Downloaded by guest on September 28, 2021 his3⌬200 leu2–3,112 ura3–52 lys2–801 trp1–1 doa4⌬1::LEU2) and inactivated (15 min, 60°C) K28 toxin⅐mlϪ1. After this incubation, its isogenic wild-type strain MHY501, both originally derived from cells were lysed (see above), cell debris and membranes were the yeast strain collection of Mark Hochstrasser (33). removed by centrifugation at 13,000 ϫ g, and the resulting super- natant applied onto a TALONspin column that had been preequili- Escherichia coli Strains, , and DNA Manipulations. Standard brated and purified according to the instructions of the manufac- molecular manipulations were performed as described by Sam- turer (Clontech). In brief, His-tagged proteins were allowed to bind brook et al. (34). For cloning, E. coli strain DH5␣ (FϪ recA1 endA1 to the matrix (5 min; 20°C), and the column resin was then washed gyr A96 thi hsdR17 supE44 relA1 ⌬(argF-lac-ZYA) U169 (⌽80 three times (50 mM sodium phosphate and 300 mM sodium dlacZ⌬M15) ␭Ϫ) was grown at 37°C in LB-medium supplemented chloride, pH 7.0) before bound proteins were eluted by the addition with 100 ␮g of ampicillin⅐mlϪ1 when necessary. PCR amplifications of elution buffer (wash buffer containing 150 mM imidazole, pH were performed by using high-fidelity TaqDNA-polymerase 7.0). Eluted proteins were subsequently concentrated by ethanol- (Roche) according to the instructions of the manufacturer and precipitation and used for Western blotting. products routinely sequenced. K28[␣͞␤], K28[␣͞␥K1͞␤] and K28[SPK1] (the latter containing the prepro-sequence from K1 Immunoblot Analysis. Protein samples were electrophoretically sep- pptox) were constructed by PCR-mediated overlap extension (35, arated in Tris⅐tricine SDS polyacrylamide gels (37). After electro- 36). After PCR amplification, the corresponding DNA fragments transfer of the proteins to a poly(vinylidene difluoride) membrane, were directly cloned into pYES2.1͞V5-HIS-TOPO according to blots were incubated with a polyclonal antibody directed against the the instructions of the manufacturer (Invitrogen), allowing regu- ␤ subunit of K28 toxin (14). Alternatively, blots were probed with lated pptox expression under transcriptional control of the galac- a monoclonal antibody directed against either a C-terminal His-tag tose-inducible GAL1 promoter. Overexpression of mutated Ub (Invitrogen) or a polyclonal anti-Ub antibody (Sigma). Invertase (Ub-RR48/63) was achieved by cotransformation of K28 pptox- (Suc2p) secretion was determined in cell-free culture supernatants ϩ expressing wild-type cells with pWO21, containing the of Suc2 wild-type strains and the ⌬suc2 mutant SEY6210, which mutated Ub gene under transcriptional control of the Cu2ϩ- had been included as negative control. In each case, aliquots of the inducible CUP1 promoter. The K294R substitution in K28-␤ (␣͞ cell-free culture supernatants were separated by SDS͞PAGE and ␤K-0) was introduced by splicing by overlapping extension (SOE)- probed with polyclonal anti-Suc2p (kindly provided by Ludwig PCR (see above) by using the K28 pptox-encoding plasmid pPGK- Lehle, Regensburg University, Regensburg, Germany). M28-I as template (29) and oligonucleotides K294Rrev and E28 as primers. The resulting PCR fragment was cloned into the yeast Killer Assay and Immunity Tests. K28-specific immunity was deter- expression vector pYX242 (Invitrogen). The lysine-free K28 variant mined in an agar diffusion assay on methylene blue agar plates ␣K-0͞␤K-0 and the pptox variant lacking any internal lysyl residue (MBA) (pH 4.7) as described in ref. 19. Toxin sensitivity͞immunity (pptoxK-0) were PCR amplified (all primers used throughout this tests were performed on MBA plates by using an overlay (105 cells study are shown in Table 1, which is published as supporting per plate) of the toxin-sensitive strain S. cerevisiae SEY6210 car- information on the PNAS web site). rying the indicated plasmid; a cell-free concentrated culture super- natant of a K28 killer strain was used as the toxin source. Briefly, Yeast Cell Fractionation. For subcellular toxin-localization, either concentrated culture supernatants (100 ␮l) were pipetted into wells the K28-susceptible strain S. cerevisiae SEY6210 or the K28- (10-mm diameter) cut into the agar, and plates were then incubated secreting superkiller (ski2–2) mutant MS300b and its heat-cured for 4 days at 20°C. In this assay, yeast transformants expressing a nonkiller derivative (19) were grown at 30°C in yeast extract͞ pptox derivative that is incapable of conferring immunity show a peptone͞dextrose medium to early exponential phase, harvested by sensitive nonkiller phenotype, resulting in a zone of growth inhi- centrifugation, and used for cell fractionation experiments essen- bition around the well. tially as described in ref. 14. We thank Charles P. Cartwright for valuable comments and for critically Purification of His-Tagged pptox. Transformants expressing a His- reading the manuscript; Mark Hochstrasser (Yale University, New Haven, CT) and Dieter Wolf for providing various yeast mutants (⌬doa4, pre1–1, tagged K28 pptox derivative lacking its N-terminal prepro sequence ⌬ and pre2–2) and plasmid pWO21; Randy Schekman and Ludwig Lehle for [ tox(His)6] were grown at 30°C to early exponential phase in kindly providing aliquots of polyclonal anti-Sec61p, anti-Kar2p, and anti- synthetic complete medium lacking uracil and then spheroplasted Suc2p antiserum; and Beate Schmitt and Nadine Laßotta for technical as described in ref. 11. Spheroplasts were subsequently incubated assistance. This work was kindly supported by Deutsche Forschungsgemein- for1hinthepresence of 104 units of either native or heat- schaft Grants Schm 541͞11-2 and Graduiertenkolleg 845-1.

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