The Ubiquitin Moiety of Ubi1 Is Required for Productive Expression of Ribosomal Protein eL40 in Saccharomyces cerevisiae

cells Article The Ubiquitin Moiety of Ubi1 Is Required for Productive Expression of Ribosomal Protein eL40 in Saccharomyces cerevisiae Sara Martín-Villanueva 1,2, Antonio Fernández-Pevida 1,2, Dieter Kressler 3,* and Jesús de la Cruz 1,2,* 1Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocio/CSIC/Universidad de Sevilla, E-41013 Seville, Spain 2Departamento de Genética, Universidad de Sevilla, E-41012 Seville, Spain 3Unit of Biochemistry, Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland *Correspondence: [email protected] (D.K.); [email protected] (J.d.l.C.); Tel.: +41-26-300-86-45 (D.K.); +34-955-923-126 (J.d.l.C.) Received: 19 July 2019; Accepted: 6 August 2019; Published: 7 August 2019   Abstract: Ubiquitin is a highly conserved small eukaryotic protein. It is generated by proteolytic cleavage of precursor proteins in which it is fused either to itself, constituting a polyubiquitin precursor of head-to-tail monomers, or as a single N-terminal moiety to ribosomal proteins. Understanding the role of the ubiquitin fused to ribosomal proteins becomes relevant, as these proteins are practically invariablyeS31andeL40inthedifferent eukaryotes. Herein, we usedtheamenableyeastSaccharomyces cerevisiae to study whether ubiquitin facilitates the expression of the fused eL40 (Ubi1 and Ubi2 precursors) and eS31 (Ubi3 precursor) ribosomal proteins. We have analyzed the phenotypic effects of a genomic ubi1 ∆ ub-HA ubi2 ∆ mutant, which expresses a ubiquitin-free HA-tagged eL40A protein as the sole source of cellular eL40. This mutant shows a severe slow-growth phenotype, which could be fully suppressed by increased dosage of the ubi1 ∆ ub-HA allele, or partially by the replacement of ubiquitin by the ubiquitin-like Smt3 protein. While expression levels of eL40A-HA from ubi1 ∆ ub-HA are low, eL40A is produced practically at normal levels from the Smt3-S-eL40A-HA precursor. Finally, we observed enhanced aggregation of eS31-HA when derived from a Ubi3 ∆ ub-HA precursor and reduced aggregation of eL40A-HA when expressed from a Smt3-S-eL40A-HA precursor. We conclude that ubiquitin might serve as a cis-acting molecular chaperone that assists in the folding and synthesis of the fused eL40 and eS31 ribosomal proteins. Keywords: ribosome biogenesis; pre-rRNA processing; ribosomal protein L40 (eL40); ubiquitin; UBI1/2 genes; translation; yeast 1. Introduction Ubiquitin is a small eukaryotic protein of 76 amino acids whose name (i.e., it occurs ubiquitously) results from its remarkable evolutionary conservation [ 1 ]. Ubiquitin functions as a reversible post-translational modifier of proteins to regulate many different cellular processes such as DNA repair, chromatin dynamics, cell cycle regulation, membrane and protein trafficking, endocytosis, autophagy, but most notably proteasome-dependent protein degradation [ 2 – 4 ]. Normally, the conjugation of ubiquitin to other proteins involves the formation of an isopeptide bond between the α -carboxyl group of the C-terminal glycine of a ubiquitin molecule with an ε -amino group of a specific lysine residue within the target protein [ 2 , 3 ]. This process is known as ubiquitination and involves three sequential steps: activation, conjugation, and ligation, which are performed by ubiquitin-activating (E1) enzymes, Cells 2019,8, 850; doi:10.3390/cells8080850 www.mdpi.com/journal/cells Cells 2019,8, 850 2 of 20 ubiquitin-conjugating (E2) enzymes, and ubiquitin ligases (E3), respectively. The reversal of this modification, deubiquitination, is catalyzed by specific proteases called deubiquitinating enzymes [ 5 ]. In most eukaryotes studied, ubiquitin is encoded by two classes of genes: (i) The first comprises genes coding for a single copy of ubiquitin fused to ribosomal proteins (r-proteins), most commonly eL40 and eS31. Ubiquitin fusion to other r-proteins, such as P1 or P2, and even to non-ribosomal proteins such as actin, has been reported in diverse genera of rare single-celled algae as, for example, Bigelowiella [ 6 ]. (ii) The second class includes genes that encode a polyubiquitin precursor protein, which consists of a polymer of several tandem ubiquitin monomers (e.g., [ 7 – 10 ]). For both classes, free de novo ubiquitin is synthesized by proteolytic maturation from the corresponding precursor proteins. Only rarely, ubiquitin is encoded by monoubiquitin genes, as occurs in the protozoa Giardia lamblia and Entamoeba histolytica, or in algae as Guillardia theta and Bigelowiella natans [11–13]. In Saccharomyces cerevisiae (thereafter yeast), but also in mammals, ubiquitin is encoded by four different genes [ 14 ]. The yeast UBI4 gene (equivalent to the human UBB and UBC genes) encodes a polyubiquitin precursor protein of five head-to-tail repeats of ubiquitin. Expression of UBI4 occurs when cells enter the stationary phase or when they are exposed to diverse stresses, including heat shock, oxidative stress, exposure to DNA-damaging agents, or nutrient depletion [ 8 , 15 – 19 ]. The paralogous UBI1 and UBI2 yeast genes (equivalent to the human UBA52 gene) code for a fusion protein where a single ubiquitin moiety is fused to the 60S r-proteins eL40A and eL40B, respectively. In turn, the yeast UBI3 gene (equivalent to the human RPS27A/UBA80 gene) codes for a fusion protein consisting of a single N-terminal ubiquitin moiety and the 40S r-protein eS31 [ 8 , 14 ]. In actively growing yeast cells, most of the ubiquitin originates from these three r-protein fusion genes [ 15 ]; on the other hand, expression of UBI4 is induced when the UBI1,UBI2, and UBI3 genes are turned down, as occurs when cells are subjected to different stresses, including starvation [20]. There are other proteins that share the ubiquitin fold, and these can also be conjugated, akin to ubiquitin, via an isopeptide bond to substrate proteins in an enzymatic process that is analogous to ubiquitination (for a review, see [ 3 , 21 ]). Among these proteins, known as ubiquitin-like modifiers, SUMO (small ubiquitin-related modifier) is the most widely spread member within eukaryotes [ 21 ]. Four SUMO genes coding for different monomeric SUMO isoforms have been identified in mammals, while yeast harbors a single essential gene, SMT3, which encodes the SUMO protein Smt3 [ 22 ]. Sumoylation of target proteins regulates various cellular processes by modulating the localization or activity of the SUMO-modified substrate proteins. While polyubiquitination mainly provides a signal for proteasome-dependent degradation (e.g., [ 23 ]), sumoylation seems to enhance the stability of the target proteins (reviewed in [ 22 , 24 ]). This latter feature has been biotechnologically exploited to increase the yield of recombinant proteins in Escherichia coli; thus, the N-terminal fusion of a single SUMO moiety to recombinant proteins, which fail to properly fold and precipitate as inclusion bodies, may significantly improve their stability and solubility (e.g., [ 25 , 26 ]). Similarly, expression of recombinant proteins containing an N-terminal ubiquitin moiety has been found to augment their yield and solubility [ 27 – 29 ]. However, whether an N-terminally attached SUMO or ubiquitin moiety could act in vivo as a physiological cis-acting chaperone for proper folding and efficient expression of the fused proteins remains to be determined (see discussion in [30]). We are interested in understanding the contribution of the ubiquitin moiety within the Ubi1/2 and Ubi3 precursors to ribosome biogenesis and function. Experimental evidence indicates that while eS31 is a quasi-essential r-protein that assembles in the nucleus, most likely into early 90S pre-ribosomal particles [ 31 , 32 ], eL40 is an essential r-protein that associates in the cytoplasm with late pre-60S r-particles [ 33 , 34 ]. Under wild-type conditions, the Ubi1/2 and Ubi3 precursor proteins could so far never be detected, suggesting that their proteolytic maturation occurs very rapidly, likely co-translationally, and consequently before the assembly of the respective r-proteins into pre-ribosomal particles [ 14 , 31 , 35 ]. We have previously studied the consequences of introducing mutations in the intersection between ubiquitin and eS31 within the Ubi3 precursor and between ubiquitin and eL40 within the Ubi1 precursor that partially or totally impair ubiquitin removal [ 35 , 36 ]. Cells 2019,8, 850 3 of 20 The obtained results indicate that the presence of ubiquitin hinders the assembly of the respective r-proteins into pre-ribosomal particles; therefore, assembly of either eS31 or eL40A is favored over that of non-cleaved Ubi3 or Ubi1, respectively [ 35 , 36 ]. Moreover, non-cleaved Ubi3 or Ubi1 variants confer lethal phenotypes when expressed as the sole source of eS31 or eL40, respectively. Only in these circumstances (i.e., upon depletion of the wild-type eS31 or eL40 counterparts), these mutant proteins get incorporated into nascent pre-ribosomal particles [ 35 , 36 ]. Strikingly, while Ubi3-containing r-particles can quite efficiently engage in translation [ 35 ], Ubi1-containing r-particles are delayed in their cytoplasmic maturation (i.e., Tif6 recycling), prevent r-subunit joining, and interfere with translation elongation [ 36 ]. In addition, we and others have shown that the ubiquitin moiety of the Ubi3 precursor can be deleted (ubi3 ∆ ub allele) without causing a deleterious phenotype as long as the unfused eS31 r-protein is expressed at an elevated dosage [ 14 , 35 ]. In contrast, expression of eS31 from an ubi3 ∆ ub allele integrated into its natural chromosomal locus conferred a pronounced slow-growth (sg) phenotype and a shortage of 40S r-subunits [ 14 , 35 ]. Thus, the N-terminal ubiquitin moiety of Ubi3 contributes to the efficient synthesis of eS31 and, as a consequence, is required for the quantitative synthesis of 40S r-subunits. In this study, we have performed an equivalent analysis with a ubi1 ∆ ub allele, which encodes a ubiquitin-free Ubi1 ∆ ub protein. As above, when the sole cellular source of eL40 originates from a single-copy ubi1 ∆ ub allele, integrated at the native UBI1 genomic locus, cells showed a pronounced sg phenotype and a deficit in 60S r-subunits; both defects are fully suppressed by increasing the gene dosage of the ubi1 ∆ ub allele by placing it on a centromeric (CEN) plasmid. Therefore, as for Ubi3, it appears that the ubiquitin moiety of the Ubi1 precursor is important for the efficient accumulation of the derived eL40A r-protein product. Importantly, expression of an N-terminally Smt3-fused eL40A variant protein as sole source of eL40 leads to a modest yet significant improvement of cell growth, especially at high temperatures. Finally, we have evaluated the effect of the absence and presence of an N-terminal ubiquitin or Smt3 moiety, respectively, on the aggregation status of HA-tagged eL40A, either expressed from a genomic UBI1-HA, ubi1 ∆ ub-HA or SMT3-S-eL40A-HA allele, and HA-tagged eS31, expressed either from a genomic UBI3-HA or ubi3 ∆ ub-HA allele. While we found practically no differences in the extent of aggregation for HA-tagged eL40A derived from the ubiquitin-containing Ubi1-HA precursor or the ubiquitin-free Ubi1 ∆ ub-HA protein in cells encompassing a ubi2 ∆ mutation, we observed both a clear tendency for reduced aggregation of eL40A-HA when generated from the Smt3-S-eL40A-HA precursor and for enhanced aggregation of HA-tagged eS31 derived from the ubi3 ∆ ub-HA allele when compared to eS31-HA originating from the UBI3-HA allele. We discuss our results in the context of a possible cis-acting role of the N-terminal ubiquitin moiety, fused to eS31 and eL40, as a molecular chaperone that facilitates the correct folding and efficient synthesis of these two r-proteins. 2. Materials and Methods 2.1. Strains and Microbiological Methods All yeast strains used in this study are listed in Table 1and are derived from the diploid W303 strain [ 37 ]. A two-step allele replacement method was used to replace genomic wild-type UBI1 by the UBI1-HA, ubi1 ∆ ub-HA or SMT3-S-eL40-HA alleles [ 38 ]. Briefly, SMY106, which is a haploid ubi1::klURA3 (URA3 gene from Kluyveromyces lactis) null mutant strain, was co-transformed with 100 ng of empty YCplac111 vector and 1 µ g of DNA fragments containing the UBI1-HA, ubi1 ∆ ub-HA, or SMT3-S-eL40-HA alleles excised by EcoRI/HindIII digestion from YCplac111-UBI1-HA, YCplac111-ubi1 ∆ ub-HA, or YCplac111-SMT3-S-eL40-HA, respectively. Transformants were selected on SD-Leu plates. Then, transformants were replica-plated onto 5-FOA-containing plates to select for those that had lost the klURA3 marker as a consequence of a site-specific recombination event. Candidate clones were analyzed by colony PCR and sequencing. SMY113 (UBI1-HA), SMY107 (ubi1 ∆ ub-HA), and SMY218 (SMT3-S-eL40A-HA) are representative 5-FOA-resistant clones that were selected for further analyses. To obtain strains where the above alleles are the sole source of cellular eL40, SMY113, Cells 2019,8, 850 4 of 20 SMY107, and SMY218 were crossed to JDY923, which is a ubi2::kanMX4 null mutant. The resulting diploids were sporulated and tetrads were dissected. Several complete tetrads from each cross were analyzed for (i) G418 resistance on G418-containing plates, (ii) the presence of the ubi2::kanMX4 deletion disruption and either the UBI1-HA, ubi1 ∆ ub-HA or SMT3-S-eL40A-HA allele by PCR, and (iii) the expression of the HA epitope by immunoblotting using anti-HA antibodies. SMY256 (UBI1-HA ubi2 ∆ ), SMY257 (ubi1 ∆ ub-HA ubi2 ∆ ), and SMY258 (SMT3-S-eL40A-HA ubi2 ∆ ) are representative meiotic segregants that were selected for further analyses. Strains SMY215 and SMY216 were generated by transforming the UBI1/2shuffle strain (TAY001) with the plasmids YCplac111-ubi1 ∆ ub-HA or YCplac111-SMT3-S-eL40-HA; transformants were then restreaked on 5-FOA-containing plates to counter-select against the presence of pHT4467∆-UBI1. Yeast cells were grown at the indicated temperatures either in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or SD medium (synthetic dextrose; 0.15% yeast nitrogen base, 0.5% ammonium sulphate, and 2% glucose), which was supplemented with the appropriate amino acids and bases as nutritional requirements. To prepare plates, 2% agar was added to the media before sterilization. Yeast genetic techniques and growth media have been previously described [ 39 ]. Yeast cells were transformed by the lithium acetate method [ 40 ]. For tetrad dissection, a Singer MSM 400 micromanipulator was used. Standard molecular biology techniques were carried out according to the specifications in Sambrook et al. [ 41 ]. E. coli DH5 α was used for cloning and propagation of plasmids [41]. Table 1. Yeast strains used in this study. Strain aRelevant Genotype bSource W303-1A MATaade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 [37] W303-1B As W303-1A but MATα[37] TAY001 MATaubi1::kanMX4 ubi2::kanMX4 ade3::kanMX4 [pHT4467∆-UBI1] [36] SMY106 MATaubi1::klURA3 This study SMY113 MATaUBI1-HA This study SMY256 MATaUBI1-HA ubi2::kanMX4 This study SMY107 MATaubi1∆ub-HA This study SMY257 MATαubi1∆ub-HA ubi2::kanMX4 This study SMY215 MATaubi1::kanMX4 ubi2::kanMX4 ade3::kanMX4 [YCplac111-ubi1∆ub-HA] This study JDY923 MATαubi2::kanMX4 [33] SMY218 MATaSMT3-S-eL40A-HA This study SMY258 MATαSMT3-S-eL40A-HA ubi2::kanMX4 This study SMY216 MATaubi1::kanMX4 ubi2::kanMX4 ade3::kanMX4 [YCplac111-SMT3-S-eL40A-HA] This study JDY532 MATassb1::HIS3MX6 ssb2::natNT2 This study SMY324 MATassb1::HIS3MX6 ssb2::natNT2 UBI3-HA::kanMX4 This study TLY56.D3 MATaUBI3-HA::kanMX4 [35] TLY61.A2 MATaubi3∆ub-HA::kanMX4 [35] TLY14.3C MATαubi3::HIS3MX6 [35] SMY315 MATαrps12::kanMX4 This study ORY211 MATαrpl39::natNT2 This study MS157-1A MATαdob1-1 [42] a Strains used in this study are isogenic with W303. b For simplicity, only the relevant genotypes, differing from the one of the W303-1A strain, are indicated. 2.2. Plasmids Plasmids used in this study were generated by multiple sequential cloning steps and they are listed in Table 2. All constructs were verified by DNA sequencing. Description of the oligonucleotides used for the PCRs and information on the construction of the different plasmids will be available upon request. Cells 2019,8, 850 5 of 20 Table 2. Plasmids used in this study. Name (Collection Name) Relevant Information Source pHT4467∆-UBI1 CEN6 (instable), URA3,ADE3. Wild-type Ubi1; promoter and terminator of UBI1 [36] YCplac111 CEN,LEU2 [43] YCplac111-UBI1-HA (pDK4131) CEN,LEU2. C-terminally 1xHA-tagged eL40A; promoter and terminator of UBI1 [36] YCplac111-ubi1∆ub-HA (pDK4192) CEN,LEU2. Allele ubi1 ∆ ub-HA (M-II); promoter and terminator of UBI1 This study YCplac111-SMT3-S-eL40-HA (pDK4193) CEN,LEU2. Allele SMT3-S-eL40A-HA (GG-S-II); promoter and terminator of UBI1 This study pADH111-Ub (pDK2253) CEN,LEU2. Wild-type ubiquitin; promoter and terminator of ADH1 This study 2.3. Polysome Analysis and Sucrose Gradient Fractionation Cell extracts for polysome analysis were prepared and analyzed as previously described [ 44 , 45 ]. Ten A 260 units of each cell extract were loaded onto 7–50% sucrose gradients. These gradients were centrifuged at 39,000 rpm in a Beckman Coulter SW41Ti rotor for 2 h 45 min; the A 254 was continuously monitored using an ISCO UA-6 system. When needed, fractions of 0.5 mL were collected and proteins were precipitated from each fraction using trichloroacetic acid, as previously described [ 46 ]. The precipitated fractions were resuspended in 2 × Laemmli loading buffer; an equal volume of each fraction was separated by SDS-PAGE and analyzed by western blotting. 2.4. Western Blot Analysis and Antibodies Total yeast protein extracts were prepared by the alkaline lysis method of Yaffe and Schatz [ 47 ], which immediately freezes the in-vivo protein content, thus preventing rapid protein turnover reactions. Proteins were separated by SDS-PAGE and analyzed by western blotting according to standard procedures [ 41 ]. The following primary antibodies were used: mouse monoclonal anti-HA 16B12 (Covance, Emeryville, CA, USA), anti-Pgk1 (Invitrogen, Eugene, OR, USA), and anti-uL3 (gift from J.R. Warner) [ 48 ]; rabbit polyclonal anti-uL29 (gift from M. Seedorf) [ 49 ] and anti-uS3 (gift from M. Seedorf) [ 49 ]. Goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Bio-Rad, Hercules, CA, USA) were used as secondary antibodies. Immune complexes were visualized using a chemiluminescencedetectionkit(Super-Signal West Pico, Pierce, Rockford, IL,USA)andaChemiDoc TM MP imaging system (Bio-Rad, Hercules, CA, USA). 2.5. Analysis of Aggregated Proteins Isolation of aggregated proteins was done from whole cell extracts as described by Koplin et al. [ 50 ], but following the sonication modifications described in Panasenko et al. [ 51 ]. Briefly, 40 OD 600 units of cells, grown to mid-log phase in YPD, were harvested, washed with water containing 15 mM NaN 3 , and cell pellets were first frozen in liquid N 2 and then stored at − 80 ◦ C. For preparation of cell lysates, the pellets were resuspended in 0.5 mL of freshly prepared lysis buffer (20 mM sodium phosphate, pH 6.8, 10 mM DTT, 1 mM EDTA, 0.1% Tween 20) supplemented with 1 × Complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland), 3 mg/mL zymolyase T20 (USBiological, Salem, MA, USA) and 1.25 U/mL benzonase. Samples were incubated at room temperature for 15 min, chilled on ice for 5 min, and tip sonicated (Branson sonifier 450; three times 10 s at duty cycle 40%). Upon sonication, the lysates were cleared by centrifugation at 200 × gfor 20 min at 4 ◦ C, and the supernatants were adjusted to an identical protein concentration, determined by the Bradford method, of 10.0 mg/mL. Supernatant aliquots of 20 µ L were taken and boiled with 40 µ L of SDS Laemmli sample buffer (total extract samples). Then, the supernatants were centrifuged at 16,000 × gfor 20 min at 4 ◦ C to pellet the aggregated proteins. After removing the supernatants, the pellets were washed twice with 0.5 mL of wash buffer (20 mM sodium phosphate, pH 6.8, 1 × Complete EDTA-free protease inhibitor cocktail) Cells 2019,8, 850 6 of 20 containing 2% NP-40 (WB-NP); then, pellets were resuspended in 0.5 mL WB-NP, sonicated (10 s at duty cycle 40%), and centrifuged at 16,000 × gfor 20 min at 4 ◦ C. Pellets were again washed twice with 0.5 mL of wash buffer and then resuspended in wash buffer, sonicated, and centrifuged as above. Pellets were resuspended in 50 µ L of SDS Laemmli sample buffer and boiled (protein aggregate samples). Total extract and protein aggregate samples were then separated by 4–12% gradient SDS-PAGE and analyzed by colloidal blue Coomassie staining and western blotting. 2.6. Reproducibility To ensure good reproducibility, each experiment was done two or more times (biological replicates); then, each sample was also analyzed at least twice (technical replicates). Representative experiments are shown in the different figures. 3. Results 3.1. Generation of ubi1 Mutants for Phenotypic Analysis Yeast eL40 is an essential r-protein of 52 amino acids that is encoded by two independent paralogous genes, UBI1 (RPL40A; YIL148W), and UBI2 (RPL40B; YKR094C). As mentioned in the introduction, eL40 as well as the r-protein eS31 (UBI3 gene) are produced in practically all eukaryotes by proteolytic removal of the N-terminal ubiquitin moiety from the ubiquitin-fused precursor r-proteins. We previously studied the effects of impaired ubiquitin removal from the Ubi1 and Ubi3 precursors by introducing specific mutations that decrease or abolish their proteolytic maturation [35,36]. Figure 1. Schematic representation of the genomic constructs used in this study. Ubi1 consists of an N-terminal ubiquitin moiety fused to the r-protein eL40A. A peptide bond between the C-terminal glycine of ubiquitin (G76) and the N-terminal isoleucine of eL40A (I77) connects the two proteins. Relevant residues and ubi1 mutant variants thereof used in this study are also indicated. The relevant constructs are schematically depicted, and the respective allele, precursor protein, and mature protein names are indicated. The asterisk denotes that the exact nature of the mature proteins derived from these precursor proteins has not been experimentally determined; thus, the indicated mature proteins correspond to the most likely in vivo cleavage products. A single C-terminal HA tag was added for western detection of the Ubi1 and/or eL40A protein variants. To date, however, only experimental data concerning the phenotypic consequences of expressing a genomic ubi3 ∆ ub allele, which lacks the region coding for the ubiquitin moiety, are available [ 14 , 35 ]. To explore the role of the N-terminal fused ubiquitin moiety within Ubi1/2 precursors, we constructed two different mutant Ubi1 variants (Figure 1). First, we replaced the endogenous wild-type UBI1 copy with a ubi1 ∆ ub-HA allele, which harbors a deletion of the ubiquitin coding region and therefore solely expresses the eL40A r-protein part without the N-terminal ubiquitin moiety. To allow efficient translation of the construct, the cognate ATG start codon of UBI1 was kept downstream of its 5 0 untranslated region; thus, the corresponding eL40A protein starts with a N-terminal methionine that is not present in the natural, proteolytically processed eL40A. Second, we replaced the endogenous Cells 2019,8, 850 7 of 20 UBI1 allele with a SMT3-S-eL40A-HA allele where the ubiquitin coding region was exchanged with a Smt3 (hereafter SUMO) coding region. To facilitate cleavage of the SUMO moiety, we inserted a serine codon at the junction between the coding regions of Smt3 and eL40A. In both constructs, as well as in the genomic UBI1-HA wild-type control construct, we also added an in-frame C-terminal single HA epitope for western detection. 3.2. The ubi1∆ub Ubiquitin Deletion Mutant Displays a Slow-Growth Phenotype For the functional analysis of the generated mutants, we first examined the growth phenotype associated with the deletion of the ubiquitin moiety from the Ubi1 precursor (Figure 2). As eL40 is encoded by two independent paralogous genes, we performed this analysis both in the presence or the absence of a functional eL40B r-protein (wild-type UBI2 or ubi2 ∆ background, respectively). As previously described [ 33 , 36 ], the presence of the HA epitope slightly affected growth when eL40A-HA is the sole source of eL40, specially at low temperatures. In agreement, doubling times of ca. 1.8 h, 2.0 h, and 3.0 h were obtained in liquid YPD at 30 ◦ C for the wild-type W303-1A strain and the isogenic ubi2 ∆ and UBI1-HA ubi2 ∆ mutants, respectively. As also discussed [ 36 ], this is likely due to an adverse effect of the extra amino acids on the interaction environment of the C-terminus of eL40A within the ribosome. Figure 2also shows that deletion of the ubiquitin moiety has practically no effect on cell growth when the UBI2 gene is present. However, ubi1 ∆ ub-HA ubi2 ∆ mutant cells, in which all cellular eL40 is derived from the single-copy expression of a ubiquitin-free eL40A construct, showed a severe sg phenotype at all tested temperatures and divided every 5.5 h in liquid YPD at 30 ◦C. Figure 2. The genomically integrated ubi1 ∆ ub-HA allele confers a slow-growth phenotype. Growth analysis of the indicated strains, either expressing a C-terminally HA-tagged Ubi1 (UBI1-HA) or a Ubi1 ∆ ub (ubi1 ∆ ub-HA) variant protein from the UBI1 genomic locus or from a centromeric plasmid (+ ubi1 ∆ ub-HA) in a wild-type UBI2 or a ubi2 ∆ null mutant background. Strains W303-1A (Wild type), SMY113 (UBI1-HA), SMY256 (UBI1-HA ubi2∆), SMY107 (ubi1∆ub-HA), SMY257 (ubi1∆ub-HA ubi2∆), SMY215 [YCplac111-ubi1 ∆ ub-HA] (ubi1 ∆ ubi2 ∆ +ubi1 ∆ ub-HA), and JDY923 (ubi2 ∆ ) were spotted in fivefold serial dilution steps onto YPD plates, which were incubated for 4 days at 22 ◦ C or for 3 days at 30 ◦C and 37 ◦C. It has been previously shown that increased dosage of the ubi3 ∆ ub allele suppresses its growth defect [ 14 , 35 ]. Thus, to address whether a similar situation occurred upon increasing the dosage of the ubi1 ∆ ub allele, we cloned either the wild-type UBI1-HA or the mutant ubi1 ∆ ub-HA allele into YCplac111, a centromeric (CEN) low-copy-number LEU2 plasmid [ 43 ], and transformed these constructs into the UBI1/2shuffle strain TAY001 (for its genotype, see Table 1). After counter-selection against the pHT4467 ∆ -UBI1 (URA3 ADE3) plasmid on 5-FOA-containing plates, cell growth was analyzed on YPD plates. As shown in Figure 2, cells harboring the ubi1 ∆ ub-HA allele on the CEN plasmid, thus constituting the sole source of cellular eL40, grew significantly better than the ubi1 ∆ ub-HA ubi2 ∆ mutant cells, in which the ubi1 ∆ ub-HA allele is present at the genomic UBI1 locus. This is in agreement Cells 2019,8, 850 8 of 20 with previous data showing that the expression of a ubi1 ∆ ub allele from a strong promoter also complemented the lethal phenotype of a ubi1 ∆ ubi2 ∆ strain to the wild-type extent [ 14 ]. We conclude that the presence of the ubiquitin moiety is required for optimal cell growth when eL40 is expressed from a single-copy allele. 3.3. The SMT3-S-eL40A ubi2∆Mutant Displays a Slow-Growth Phenotype The small ubiquitin-like modifier SUMO has an overall three-dimensional structure that is very similar to the one of ubiquitin and possesses, as ubiquitin, a C-terminal glycine residue via which it is conjugated to specific lysine residues of target proteins (e.g., [ 52 ]). Thus, given that ubiquitin provides a beneficial cis-acting effect when fused to eL40, we reasoned that the replacement of the ubiquitin moiety in the Ubi1 precursor by SUMO should therefore also permit, if enabling proper processing of the Smt3-eL40A fusion protein by the SUMO proteases Ulp1 and Ulp2 [ 53 ], the optimal production of functional eL40A protein. To this end, we integrated the SMT3-S-eL40A-HA allele at the genomic UBI1 locus (see Section 3.1 and Materials and Methods). Then, we assessed the growth of cells harboring the SMT3-S-eL40A-HA allele in the context of the wild-type UBI2 and the ubi2 ∆ null mutant background. As shown in Figure 3,SMT3-S-eL40A-HA cells grow practically like the wild-type control strain in the presence of UBI2. Interestingly, the SMT3-S-eL40A-HA ubi2 ∆ strain, where the Smt3-S-eL40A-HA precursor is the sole source of eL40, displays a mildly improved growth compared to that of the isogenic ubi1 ∆ ub-HA ubi2 ∆ strain, which is especially evident at higher temperatures. Consistently, when grown in liquid YPD at 30 ◦ C, SMT3-S-eL40A-HA ubi2 ∆ cells divided more rapidly than ubi1 ∆ ub-HA ubi2 ∆ cells as indicated by the determined doubling times of 4.0 h and 5.5 h, respectively. As observed for the ubi1 ∆ ub-HA allele, introducing the SMT3-S-eL40A-HA allele on a low-copy-number plasmid into ubi1∆ubi2∆cells resulted in apparent wild-type growth, showing that its increased gene dosage suppressed the sg phenotype linked to the genomic single-copy SMT3-S-eL40A allele. We conclude that SUMO is able to partially fulfil the cis-acting role of ubiquitin within the Ubi1 precursor when replacing it as the N-terminal fusion moiety preceding the eL40A r-protein component. Figure 3. The genomically integrated SMT3-S-eL40A-HA allele permits better growth than the corresponding ubi1 ∆ ub-HA allele. Growth analysis of the indicated strains, either expressing a C-terminally HA-tagged Ubi1 (UBI1-HA) or a Smt3-S-eL40A (SMT3-S-eL40A-HA) fusion variant protein from the UBI1 genomic locus or from a centromeric plasmid (+SMT3-S-eL40A-HA) in a wild-type UBI2 or a ubi2 ∆ null mutant background. Strains W303-1A (Wild type), SMY218 (SMT3-S-eL40A-HA), SMY258 (SMT3-eL40A-HA ubi2 ∆ ), SMY256 (UBI1-HA ubi2 ∆ ), SMY257 (ubi1 ∆ ub-HA ubi2 ∆ ), SMY216 [YCplac111-SMT3-S-eL40A-HA] (ubi1 ∆ ubi2 ∆ +SMT3-S-eL40A-HA), and JDY923 (ubi2 ∆ ) were spotted in fivefold serial dilution steps onto YPD plates, which were incubated for 4 days at 22 ◦ C and for 3 days at 30 ◦C and 37 ◦C. 3.4. Contribution of the Ubiquitin and SUMO Moieties to the Expression of eL40A The fact that the sg phenotype linked to providing either the ubi1 ∆ ub-HA or the SMT3-S-eL40A-HA allele as the sole source of cellular eL40 r-protein was suppressed by increasing the copy number Cells 2019,8, 850 9 of 20 of these alleles strongly suggests that the initial presence of the ubiquitin moiety within the Ubi1 precursor ensures that the released eL40A r-protein is produced in sufficient amounts. To test this hypothesis, we monitored the expression levels of the eL40A-HA r-protein in the different used strains by western blotting. As shown in Figure 4, the protein levels of eL40A-HA were significantly lower in the ubi1 ∆ ub-HA ubi2 ∆ strain when compared with those of the UBI1-HA ubi2 ∆ strain. As expected, an increased dosage of the ubi1 ∆ ub-HA allele fully restores the levels of eL40A-HA (Figure 4, lane 5). Together, these results indicate that the ubiquitin moiety of Ubi1 is required for the efficient production of the eL40 r-protein. To assess the efficiency of cleavage of the Smt3-S-eL40A-HA precursor into SUMO and eL40A-HA, western blot analyses using anti-HA antibodies were also carried out with whole cell extracts of strains harboring a genomic SMT3-S-eL40A-HA allele. As previously shown [ 33 , 36 ], no Ubi1-HA precursor but only mature eL40A-HA was detected in the UBI1-HA strains (Figure 4, lanes 2 and 3). In contrast, although cell extracts of the SMT3-S-eL40A-HA ubi2 ∆ strain mostly contained mature eL40A-HA, some precursor protein could also be detected at the expected molecular mass, especially when the Smt3-S-L40A-HA fusion protein variant was the sole cellular source of eL40A-HA (Figure 4, lanes 6 and 7). We conclude that proteolytic processing of the Smt3-S-L40A-HA precursor into SUMO and eL40-HA is less efficient than removal of ubiquitin from the Ubi1 precursor. Nevertheless, we observed a clear increase in the total amount of produced mature eL40A-HA in the cell extracts of the SMT3-S-eL40A-HA ubi2 ∆ strain when compared to the eL40A-HA levels present in the ubi1 ∆ ub-HA ubi2∆strain. Figure 4. Analysis of the protein levels of eL40A-HA. A wild-type control strain (lane 1) and strains with the indicated relevant genotypes, expressing C-terminally HA-tagged eL40A from the different precursor constructs, either from the genomic locus (lanes 2 , 3 , 4 , 6 , and 7 ) or from a centromeric plasmid (lane 5 ), in a wild type UBI2 or a ubi2 ∆ null mutant background, were grown in liquid YPD medium at 30 ◦ C. Total cell extracts were prepared and subjected to western analysis using anti-HA and anti-Pgk1 (loading control) antibodies. The mature eL40A-HA and the Smt3-S-eL40A-HA precursor are indicated. 3.5. The Genomic ubi1∆ub-HA and SMT3-S-eL40A-HA Alleles Affect the Functional Integrity of 60S r-Subunits Asthegenomicubi1 ∆ ub-HAallelelimitstheproduction of the eL40A proteininaubi2 ∆ background (Figure 4), we wondered whether this would affect the synthesis and function of 60S r-subunits. To answer this question, we performed polysome profile analyses with cell extracts obtained from this strain and, to evaluate the effect of the SUMO fusion, the SMT3-S-eL40A-HA ubi2 ∆ strain. First, we examined the profiles of the UBI1-HA ubi2 ∆ control strain grown in YPD at 30 ◦ C. This strain, in agreement with its slightly slower growth (Figure 2), still displayed good amounts of actively translating ribosomes, as inferred from the overall polysome content (Figure 5A). However, compared Cells 2019,8, 850 16 of 20 cells, ubiquitin cleaved from UBA52 has been suggested to form a complex with eL40 for regulation of protein synthesis [ 59 ]; however, this complex has not been shown to facilitate eL40 assembly. (ii) A more plausible scenario proposes that ubiquitin fulfils a role as a cis-acting chaperone to facilitate translation and folding of eL40 and eS31. While only the correctly folded r-proteins are competent for assembly into pre-ribosomal particles, the misfolded fraction of these will have an increased propensity for aggregation and/or be cleared by degradation. In agreement with such a cis-acting role of ubiquitin, we show that the expression of ubiquitin-free eL40A is clearly reduced (Figure 4) and that the replacement of the ubiquitin moiety of Ubi1 by the ubiquitin-like protein SUMO (yeast Smt3), which is known to increase the productive expression of fused proteins [ 27 – 29 , 65 ], is able to partially suppress the growth defect of the ubi1 ∆ ub-HA ubi2 ∆ mutant, especially at high temperatures (Figure 3), and leads to higher yields of the mature eL40A-HA protein (Figure 4). The fact that the Smt3-S-eL40A-HA precursor is not efficiently processed (Figures 4and 6) could be the reason for the incomplete suppression, as ubiquitin release from eL40 is required for the cytoplasmic maturation and proper function of 60S r-subunits [ 36 ] (see also Figures 5and 6). Additional experiments are required to understand the reasons why processing of the Smt3-S-eL40A-HA precursor is inefficient, although it could be that the two SUMO proteases Ulp1 and Ulp2, which are presumed to cleave this fusion protein, might be limiting as they are localized in the nuclear periphery and in the nucleus, respectively (reviewed in [ 53 ]). Moreover, to further explore the role of ubiquitin as a chaperone of r-proteins eL40 and eS31, we have analyzed the aggregation status of different eL40 and eS31 protein variants. In general, most r-proteins have a high tendency to aggregate during or after their synthesis due to their particular characteristics, such as an extremely high isoelectric point and the presence of unstructured or intrinsically disordered extensions [ 66 ]. These features have spurred the evolution of eukaryote-specific proteins known as dedicated chaperones and escortins, which facilitate the import and/or assembly of individual r-proteins, thus impeding their degradation, inappropriate interaction with other cellular components, and aggregation [ 67 – 69 ]. Additionally, r-proteins are among the major client proteins of the general ribosome-associated chaperones such as the nascent polypeptide-associated complex (NAC) and the Ssb-RAC chaperone triad, consisting of the Hsp70 chaperone Ssb and the ribosome-associated complex (RAC) [ 50 ]. It has been shown that the loss of NAC and Ssb-RAC complexes causes aggregation of most r-proteins from both r-subunits [ 50 ] (see also Figures 8and 9). Although originally r-proteins eL40 and eS31 were not found among the proteins identified in the aggregation analysis of cells lacking NAC and Ssb-RAC (see [ 50 ]), our results show that, at least, the fused ubiquitin moiety does not prevent the specific aggregation of eS31 in the absence of the Ssb chaperone (Figure 9). Interestingly, deleting the ubiquitin moiety from the Ubi3 precursor causes a mild enhancement of the aggregation levels of eS31; however, this enhancement is not restricted to eS31 but is also observed for other directly tested r-proteins (uL29 and uS3, see Figure 9). Indeed, we show herein that a defect in ribosome biogenesis (e.g., analysis of the ubi3 ∆ , rps12 ∆ , rpl39 ∆ or dob1-1 mutants), similarly to what is observed in cells lacking Ssb, leads to a substantial increase in the amounts of insoluble proteins detected by polyacrylamide gel electrophoresis and Coomassie staining (Figures 8and 9). While this work was in progress, Tye et al. demonstrated that an imbalance in the synthesis of r-proteins and rRNAs leads to the rapid aggregation of newly synthesized r-proteins [ 57 ]. Thus, it is likely that the modest increase in overall aggregation detected in ubi3 ∆ ub cells could also be due to the ribosome biogenesis defect caused by the ubi3 ∆ ub allele, which impairs 40S r-subunit synthesis [ 35 ]. Similarly, the UBI1-HA ubi2 ∆ strain showed a substantial amount of insoluble material and aggregation of r-proteins, as both the ubi2 ∆ mutation and, to a lesser extent, the C-terminal HA-tag on Ubi1 interfere with ribosome biogenesis and cell growth [ 33 , 36 ] (see also Figures 2and 5A). Unfortunately, this general aggregation appears to be sufficient to mask the possible specific aggregation of eL40A-HA due to its expression from the ubiquitin-free ubi1 ∆ ub allele in ubi2 ∆ cells. Notably however, our results indicate that the fusion of Smt3 to eL40 seems to modestly prevent the aggregation of the processed r-protein. Altogether, these experiments, although not fully conclusive, strongly suggest that the ubiquitin moiety could indeed facilitate in cis the productive expression and proper folding of the fused eL40 and eS31 r-proteins. Cells 2019,8, 850 17 of 20 5. Conclusions The use of amenable tools in yeast biochemistry and genetics has enabled us to shed light on the biological role of ubiquitin fused to the r-proteins eL40 and eS31. Our findings provide evidence for how ubiquitin could contribute to the efficient expression and folding of its fused r-protein tails, eL40 and eS31, in order to furnish them as assembly-competent and fully functional mature r-proteins. With the exception of a ubiquitin variant that remains attached to eS31 in the protozoan parasite G. lamblia, there is so far no evidence of a naturally occurring non-cleaved form of ubiquitin fused to either eS31 or eL40 as a constituent of mature ribosomes [ 70 ]; thus, proteolytic removal of the ubiquitin moiety from the precursors seems to be a general requirement for the functionality of the r-subunits containing these two r-proteins. Furthermore, our results suggest that the fused ubiquitin moiety may act as an in-vivo chaperone enabling the efficient synthesis of eL40 and eS31, a function that can, although only partially, also be artificially fulfilled by the yeast ubiquitin-like Smt3 protein. In support of such a chaperone function, ubiquitin fused to eS31 has indeed evolutionarily diverged into a ubiquitin-like moiety in some organisms, such as, for example, in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae [71]. Author Contributions: Conceptualization, D.K. and J.d.l.C.; Investigation, S.M.-V., A.F.-P., D.K. and J.d.l.C.; Methodology, S.M.-V., A.F.-P., D.K. and J.d.l.C.; Validation, S.M.-V., A.F.-P., D.K. and J.d.l.C.; Funding acquisition, D.K. and J.d.l.C.; Project administration, D.K. and J.d.l.C.; Resources, D.K. and J.d.l.C.; Writing—Original draft preparation, S.M.-V., A.F.-P., D.K. and J.d.l.C.; Writing—Review and editing, D.K. and J.d.l.C. Funding: This research was funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) and the ERDF (BFU2016-75352-P AEI/FEDER, EU) to J.d.l.C and from the Swiss National Science Foundation (31003A_156764 and 31003A_175547) to D.K. S.M.-V. is the recipient of a FPI contract from MINECO. A.F.-P. was recipient of a fellowship from the University of Seville and a bridge contract from the Internal Research Plan from the University of Seville. 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