Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells

Abstract In eukaryotic organisms, Elongator is a six‐subunit protein complex required for the formation of 5‐carbamoylmethyl (ncm5) and 5‐methylcarboxymethyl (mcm5) side chains on uridines present at the wobble position (U34) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in‐frame 5′ AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full‐length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC‐MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full‐length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N‐terminal truncation of Elp1p is not likely to regulate Elongator complex activity.


Introduction
The Elongator complex of Saccharomyces cerevisiae was first reported to be associated with the hyper phosphorylated elongating form of RNA polymerase II (Pol II) and three proteins (Elp1p, Elp2p, and Elp3p) constituted the identified complex ). Subsequently, Elp4, Elp5, and Elp6 were identified to be a subcomplex of Elongator complex (Krogan and Greenblatt 2001;Li et al. 2001;Winkler et al. 2001). Initially, the complex was suggested to be involved in elongation of Pol II transcription through histone H3 and H4 acetylation (Wittschieben et al. 1999). Additional studies reported a role of Elongator in other cellular processes, that is, polarized exocytosis (Rahl et al. 2005), DNA repair , and formation of 5-methoxycarbonylmethyl (mcm 5 ) or 5-carbamoylmethyl (ncm 5 ) side chains at the wobble position (U 34 ) .
In yeast, 11 tRNA species have a mcm 5 or ncm 5 side chains at the wobble position and three of these species, tRNA Lys mcm 5 s 2 UUU , tRNA Gln mcm 5 s 2 UUG , and tRNA Glu mcm 5 s 2 UUC are also modified with a 2-thio group, generating the modified nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U) (Smith et al. 1973;Kobayashi et al. 1974;Kuntzel et al.

ORIGINAL RESEARCH
Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells 1975;Yamamoto et al. 1985;Keith et al. 1990;Glasser et al. 1992;Huang et al. 2005;Lu et al. 2005;Johansson et al. 2008). Overexpression of various combinations of hypomodified tRNA Lys s 2 UUU , tRNA Gln s 2 UUG , and tRNA Glu s 2 UUC suppresses the Elongator-dependent phenotypes in Pol II transcription, exocytosis, and DNA repair, but not the tRNA modification defect (Esberg et al. 2006;Chen et al. 2011). Thus, the physiological relevant function of Elongator complex in yeast is in formation of mcm 5 and ncm 5 side chains at U 34 of tRNA (Esberg et al. 2006;Chen et al. 2011). This hypothesis was recently supported by the observation that MinElp3, the homolog of yeast Elp3p in the archaea Methanocaldococcus infernus, produced cm 5 U in the presence of SAM (S-adenosyl methionine) and acetyl-CoA (Selvadurai et al. 2014). As presence of wobble uridine modifications are important for efficient translation in yeast, the pleiotropic phenotypes of mutants deficient in wobble uridine modifications seem to be caused by a defect in translation (Esberg et al. 2006;Björk et al. 2007;Dewez et al. 2008;Johansson et al. 2008;Nakai et al. 2008;Schlieker et al. 2008;Leidel et al. 2009;Chen et al. 2011;Bauer and Hermand 2012;Rezgui et al. 2013;Zinshteyn and Gilbert 2013).
Elp1p, the largest subunit of the Elongator complex, is a phosphoprotein and its dephosphorylation was dependent on the phosphatase Sit4p and its associated partners -Sap185p and Sap190p (Jablonowski et al. 2004). In a sit4 null mutant, Elp1p is hyperphosphorylated, whereas in the casein kinase hrr25 null mutant, Elp1p was hypophosphorylated (Mehlgarten et al. 2009). The proportion of hyper-and hypophosphorylated Elp1p is balanced in wild-type cells and any changes that perturb this equilibrium was suggested to result in inactivation of the Elongator (Mehlgarten et al. 2009). Therefore, Sit4p and Hrr25p seem to regulate the phosphorylation status of Elp1p and play antagonistic roles in the function of the Elongator complex (Mehlgarten et al. 2009). In a recent study, nine in vivo phosphorylation sites within Elp1p were identified and Hrr25p directly phosphorylates two of them (Ser-1198 andSer-1202) (Abdel-Fattah et al. 2015). These authors concluded that Elp1p phosphorylation plays a positive role in tRNA modification.
Aside from phosphorylation, Elp1p also undergoes proteolysis. Affinity purification of Elongator from a strain having a carboxy terminal tandem affinity purification (TAP) tag or western blot analysis of strains having an Elp1p tagged with human influenza hemagglutinin (HA), revealed two major and a minor form of Elp1p (Krogan and Greenblatt 2001;Fichtner et al. 2003). LC-MS (liquid chromatography mass spectrometry) analysis showed that the shortest form had an N-terminal truncation, resulting in a removal of about 200 amino acids (Fichtner et al. 2003). In mutants lacking Urm1p or Kti11p, the level of the Nterminal-truncated Elp1p increased (Fichtner et al. 2003).
The URM1 and KTI11 gene products were linked to Elongator as strains with these genes mutated as well as Elongator mutants are resistant to zymocin, a Kluyveromyces lactis toxin (Frohloff et al. 2001;Fichtner and Schaffrath 2002;Huang et al. 2008). Now it is known that the γtoxin, a subunit of zymocin, is an endonuclease that targets tRNA Glu mcm 5 s 2 UUC , tRNA Glu mcm 5 s 2 UUG , and tRNA Lys mcm 5 s 2 UUU (Lu et al. 2005). At the wobble position, these tRNAs have the modified nucleoside mcm 5 s 2 U 34 and the endonuclease cleaves these tRNAs between U 34 and U 35 provided that the wobble nucleoside is fully modified (Lu et al. 2005). The Kti11p is required for formation of the mcm 5 and Urm1p for the s 2 group of the mcm 5 s 2 U 34 nucleoside ). Loss of Urm1p or Kti11p increases the amount of truncated Elp1p, abolishes formation of mcm 5 or s 2 group of mcm 5 s 2 U 34 , and therefore makes cells resistant to γtoxin, suggesting that both Kti11p and Urm1p influenced Elp1p proteolysis and are required for proper Elongator function/ regulation (Fichtner et al. 2003). However, it was not established how the truncated form of Elp1p was generated, neither was the exact truncation site determined.
In this study, we identified the vacuolar protease Prb1p to be required for cleavage of Elp1p between its 203rd (Lys) and 204th (Ala) residues. Expression of N-terminal truncated Elp1p did not complement the wobble uridine tRNA modification defect of strain with an elp1Δ null allele. We found that appearance of N-terminal truncated Elp1p is a preparation artifact which can be circumvented using an alternative protein extraction method.
Plasmid construction, PCR mutagenesis, and overlapping PCR Plasmid pBY1767, a pRS315 derivative with a functional ELP1 gene cloned as a SalI and SacI fragment, was digested with restriction endonucleases SalI and BamHI. The BamHI is unique in the ELP1 gene and the SalI/ BamHI fragment containing the N-terminus of the ELP1 gene was cloned into the corresponding restriction sites of pRS315 generating pBY2015 (pRS315-ELP1 N-terminal ). By PCR (polymerase chain reaction)-based mutagenesis (QuikChange ® Agilent Technologies, Santa Clara, California, USA, XL Site-Directed Mutagenesis Kit, Catalog #200516), using oligonucleotides 5 ′ -C A G TA C A A AT G C C TA AT G G C T T T T G G T T GAACATGACAAGA-3′ or 5′-GGTCAAAGAGGCAGGAGCT AAGATCAAATTTGCGTAATCTTATTA-3′, the first (ATG 1 ) or the second (ATG 2 ) methionine codons of the ELP1 ORF in plasmid pBY2015 were changed to (TTG) leucine codons, generating plasmids pRS315-elp1 N-terminal (ATG1-TTG) and pRS315-elp1 N-terminal (ATG2-TTG) , respectively. Sequencing confirmed that no additional mutations than the ATG to TTG mutations were obtained during PCR mutagenesis. Correct plasmids were digested with restriction endonucleases SalI and BamHI and the fragments were cloned into plasmid pBY1767, exchanging the N-terminal region of the ELP1 gene, generating plasmids pBY2016 (pRS315-elp1 (ATG1-TTG) ) and pBY2017 (pRS315-elp1 (ATG2-TTG) ), respectively. To generate a plasmid expressing a truncated Elp1p starting at Ala204, overlapping PCR was applied. Plasmid pBY2015 (pRS315-ELP1 N-terminal ) was used as template and oligonucleotides 5′-CGAGGTCGACGCTCTCCCTT-3′ and 5′-TTACCT ACCAAACCTGATGCCATATTTGATCTTAGCTCCT-3′ were used to amplify the promotor region and the N-terminal part of ELP1 including ATG 2 . Oligonucleotides 5′-TAGTGGATCCATTTGTGATT-3′ and 5′-AGGAGCTAAG ATCAAATATCGCATCAGGTTTGGTAGGTAA-3′ were used to amplify DNA between the region of the ELP1 gene encoding amino acid 204 and the unique BamHI site. The PCR products were mixed and used as template for a second PCR to generate a DNA fragment where the translational start (ATG 2 ) is linked to the GCA-Ala codon corresponding to amino acid 204 in Elp1p. The PCR product was digested with restriction enzymes SalI/ BamHI and the fragment was used to replace the corresponding fragment of plasmid pBY1767, generating plasmid pBY2025 (pRS315-elp1 ATG2-GCA(Ala204) ). The plasmid was digested with restriction enzymes SacI and SalI and the elp1 ATG2-GCA(Ala204) containing fragment was cloned into the corresponding sites of pRS425, generating pBY2050 (pRS425-elp1 ATG2-GCA(Ala204) ). Plasmids used in this study are listed in Table 2.
tRNA isolation and high-pressure liquid chromatography analysis Approximately 2 g of cells was collected from yeast cultures grown to mid-log phase and cells were resuspended in 3 mL of 0.9% NaCl. The cell suspension was vortexed with 8 mL water-saturated phenol at room This study temperature (RT) for 30 min and vortexed for another 15 min with 400 μL chloroform. The water phase was isolated after centrifugation at 12,000g for 20 min and mixed with 4 mL phenol and vortexed for another 15 min. The water phase was collected after centrifugation at 12,000g for 20 min, mixed with 2.5 volumes 99.5% EtOH, and kept at −20°C for at least 3 h. Total tRNA was precipitated by centrifugation at 12,000g for 20 min. The pellet was dissolved in 5 mL DE52-binding buffer (0.1 mol/L Tris-HCl, pH 7.4, and 0.1 mol/L NaCl) and loaded onto a diethylaminoethyl DE52 cellulose column. The column was washed twice with 7 mL DE52-binding buffer. Total tRNA was eluted with 7 mL tRNA elution buffer (0.1 mol/L Tris-HCl, pH 7.4, and 1 mol/L NaCl) and precipitated with 5 mL isopropanol at −20°C for at least 3 h. Total tRNA was collected by a 12,000g centrifugation for 20 min and washed with 70% EtOH followed by another centrifugation at 12,000g for 20 min. The pellet was dissolved in 50 μL Milli-Q water. Isolated tRNA (~50 μg) was digested with one unit of nuclease P1 (Sigma, St. Louis, Missouri, USA) for 16 h at 37°C and treated with 0.5 units bacterial alkaline phosphatase for 2 h at 37°C. The hydrolysate was analyzed by high-pressure liquid chromatography (HPLC) using a Develosil C-30 reversephase column as described elsewhere (Björk et al. 2001).

Protein extraction and western blot
Cells were grown at 30°C to logarithmic phase (OD 600 ~0.5). For protein extraction without trichloroacetic acid (TCA), 20 OD 600 units of cells were resuspended in 700 μL breaking buffer (50 mmol/L Tris-HCl [pH 7.5], 50 mmol/L NaCl, 0.2% TritonX-100) and complete protease inhibitor cocktail (Roche Applied Science, Penzberg, Upper Bavaria, Germany, 05056489001), following the manufacturer's recommendation. Cells were broken with glass beads, and 10 μL (100 μg) of isolated total protein was loaded on SDS (sodium dodecyl sulfate) gels (Lamb et al. 1994;Fichtner et al. 2003). For protein extraction including TCA, five OD 600 units of cells were resuspended in 500 μL breaking buffer (20 mmol/L Tris-HCl [pH 8.0], 50 mmol/L NH 4 OAc, 2 mmol/L EDTA, and complete protease inhibitor cocktail), mixed with 500 μL ice-cold 20% TCA, and broken with glass beads. Cell suspension was centrifuged, dissolved in 300 μL TCA-Laemmli loading buffer (Hann and Walter 1991), and 10 μL of it was loaded on SDS gel (Peter et al. 1993). During cell lysis for both methods, a FastPrep-24 homogenizer (MP Biomedicals, Santa Ana, California, USA) was used to break the cells. Yeast anti-Elp1p antibody recognizing the carboxyl-terminus was designed based on previous work (Wittschieben et al. 1999) and obtained from GenScript. Anti-GFP antibody was obtained from Roche. A 1:1000 dilution of both antibodies were used to detect Elp1p and Elp1p-GFP, respectively. The actin and αtubulin levels were detected by using mouse anti-Act1 antibody (Thermo Scientific, Waltham, Massachusetts, USA) and rat anti-αtubulin antibody (Sigma Aldrich, St. Louis, Missouri, USA) at a 1:1000 dilution.

Protein digestion and LC-MS
Intact proteins were TMT-0 labeled (Pierce, Waltham, Massachusetts, USA) according to the manufacturer's instructions.The labeled proteins where denatured in 6 mol/L guanidine buffer and reduced with DTT (3 mg/mL) at 75°C for 60 min followed by alkylation using iodoacetamide (15 mg/mL) for 30 min in the dark at room temperature. Reduced and alkylated proteins where digested overnight with either trypsin (Promega, Madison, Wisconsin, USA) or GluC (Roche, Penzberg, Upper Bavaria, Germany) in 50 mmol/L ammonium bicarbonate buffer (pH 8.5). The peptides were cleaned using in-house produced stage tips (Rappsilber et al. 2003), dried, and resuspended in 0.1% trifluoroacetic acid for analysis by reverse phase liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Peptides where separated on a nano ACQUITY ™ UPLC system (Waters, MA) solvent A (0.1% formic acid [FA] in water), An elp1 null mutant (UMY3906) was transformed with plasmid pRS315, pRS315 with a wild-type ELP1 gene, pRS315 with an elp1 gene having ATG 1 mutated to TTG, or pRS315 with an elp1 gene having ATG 2 mutated to TTG. The expression of Elp1p was detected by anti-Elp1p antibody. Total protein was extracted using a method not including TCA (trichloroacetic acid). (C) Levels of modified nucleosides in strains described in (B). Total tRNA was isolated from three biological replicates and levels of modified nucleosides ncm 5 U, mcm 5 U, and mcm 5 s 2 U were determined by high-pressure liquid chromatography (HPLC). Pseudouridine (psi) was used as internal control. Error bars represent standard deviation from three biological replicates. N.D. indicates not detectable.
solvent B (0.1% FA in ACN, Acetonitrile) equipped with a C18 75 μm × 100 mm reverse phase column (Waters) using a gradient of 1-30% solvent B over 90 min with a flow rate of 300 nL/min. The mass spectrometer (Waters Synapt G2 HDMS) equipped with a nanoflow electrospray ionization (ESI) interface was operated in positive ionization mode with a minimal resolution of 20,000. All data were collected in continuum mode and mass-corrected using Glu-fibrinopeptide B. The data were processed with Protein Lynx Global Server v.2.5.2 (Waters) and the resulting spectra were searched against Uniprot databank with S. cerevisiae as taxonomy filter on our in-house MASCOT server (Matrix Science Ltd.), using a precursor tolerance of 10 ppm and a fragment tolerance of 0.1 Da.

Results and Discussion
Translation of Elp1p starts at the second AUG of the ELP1 ORF In the S. cerevisiae ELP1/YLR384C ORF there is an inframe ATG codon 48 nt downstream the first ATG codon (Fig. 1A). Based on comparison of closely related Saccharomyces species, the second but not the first ATG codon is conserved, suggesting that the start site of translation is the second ATG (Kellis et al. 2003). In order to analyze if ATG 1 or ATG 2 acts as the translational start codon, both codons were independently mutagenized from ATG (Met) to TTG (Leu) by oligo-directed mutagenesis. Plasmids with either mutant derivative of the ELP1 gene were transformed to an elp1 null mutant (UMY3906) and the expression of Elp1p was determined by western blot. When ATG 1 was mutated to TTG, the expression level of Elp1p was similar as wild type, whereas when ATG 2 was mutated, no Elp1p was detected (Fig. 1B). Consistent with these findings, analysis of modified nucleosides from tRNA revealed that the plasmid with ATG 1 mutated to TTG complemented the wobble uridine modification defect, and the plasmid with ATG 2 mutated to TTG did not complement the tRNA modification defect (Fig. 1C). Thus, under these growth conditions, that is, exponential growth in synthetic complete media lacking leucine, AUG 2 is the physiological translational start codon for Elp1p. , and pep4Δ (YSC1053-YPL154C) strains. Total protein was extracted using a method not including TCA (trichloroacetic acid) as described in Experimental Procedures section. Elp1p was detected using an Elp1p antibody recognizing the C-terminus of Elp1p. Single asterisk represented full-length Elp1p and double asterisk truncated Elp1p. (C) Quantification of ncm 5 U, mcm 5 U, and mcm 5 s 2 U nucleoside levels in total tRNA isolated from wild-type (BY4741) and prb1Δ mutant (YSC1053-YEL060C) strains. Analysis of nucleosides was done by high-pressure liquid chromatography (HPLC). The modification level is shown as the ratio between xm 5 U and pseudouridine (psi), where xm 5 U is ncm 5 U, mcm 5 U, or mcm 5 s 2 U. Error bars represent standard deviation from three biological replicates. The protease Prb1p is required for Nterminal truncation of Elp1p In order to analyze the in vivo composition of the Elongator complex, a TAP-tagged Elp1p was expressed in strain YSC1177-YLR384C and the complex was purified using the TAP procedure (Puig et al. 2001). Following purification, the identity of each band extracted from an SDS-PAGE gel was determined by mass spectrometry (data not shown). Consistent with an earlier TAP purification of Elongator complex, a six-subunit complex was obtained ( Fig. 2A, left panel) (Krogan and Greenblatt 2001). Also consistent with the earlier affinity purification or western blot using human influenza HA-tagged Elp1 protein, two major (~160 and ~140 kDa) and a minor form (~120 kDa) of Elp1p was observed ( Fig. 2A, left panel) ( Krogan and Greenblatt 2001;Fichtner et al. 2003). The two major forms are full-length Elp1p and an N-terminal-truncated Elp1p, whereas the minor form represents an Elp1p that is truncated at both N-and C-termini (data not shown). Elongator complex is a dimeric complex containing two copies of the six-subunit complex (Glatt et al. 2012). Therefore, the Elp1p form missing the C-terminus is most likely copurified as part of the dimeric Elongator complex. In addition to these three forms, a fourth form of Elp1p (~26 kDa) representing the Elp1p N-terminus was identified ( Fig. 2A, left panel). As the fulllength Elp1p (~160 kDa) is processed to generate ~140 and ~26 kDa fragments, we hypothesized that an endopeptidase should be responsible for the cleavage of Elp1p.
To identify the protease required for Elp1p cleavage, we screened 95 nonessential peptidase/protease null mutants strains (Table S1) for the inability to produce truncated Elp1p. The pattern of Elp1p was determined in protein extracts from the mutants by western blot, utilizing an antibody recognizing the C-terminus of Elp1p. Among the peptidase and protease mutants tested, we found that the prb1Δ and pep4Δ mutants showed no or reduced amounts of truncated Elp1p (Fig. 2B). Prb1p is a vacuolar protease with a complex maturation pathway that is dependent on Pep4p (Mechler et al. 1988;Moehle et al. 1989). From the prb1 null mutant strain (UMY3864), the Elongator complex was purified by TAP-tag affinity purification ( Fig. 2A, right  panel). In the absence of Prb1p, the ~140, ~120, and ~26 kDa fragments were not observed ( Fig. 2A, right panel). We also observed reduced amounts of the subcomplex consisting of Elp4p, Elp5p, and Elp6p. This is not caused by the prb1Δallele, rather it reflects that the TAP-tag is located on Elp1p, making the purification more efficient for the Elp1p-Elp3p core complex and occasionally the Elp4p-Elp6p subcomplex is less efficiently purified.
In order to investigate whether loss of Prb1p influences levels of modified nucleosides, total tRNA from the prb1Δ (YSC1053-YEL060C) and wild-type (BY4741) strains was isolated and the levels of ncm 5 U, mcm 5 U, and mcm 5 s 2 U nucleosides were determined. No significant difference was observed in levels of modified nucleosides (Fig. 2C). These results show that removal of Prb1p does not influence the ability of the Elongator complex to modify wobble uridines in tRNA.

Elp1p is cleaved between 203rd (Lys) and 204th (Ala) residues
A short form of Elp1p was reported to be missing in about 200 amino acids in the N-terminus (Fichtner et al. 2003). To precisely determine the Prb1p cleavage site, we purified the Elongator complex from wild-type strain (YSC1177-YLR384C). The purified Elongator complex ( Fig. 2A, left panel), containing both full-length (~160 kDa) and various truncated Elp1p fragments (~140, ~120, and ~26 kDa),  was labeled with an amine-reactive Tandem Mass Tag (TMT), which will attach to any free N-terminus and to side chains of lysines (K). The benefits of TMT tagging is twofold; first, it generates distinct mass shifts that are used when identifying the peptides and second, upon fragmentation a reporter ion is generated that helps to verify that the tag is present on the peptide. The trypsin protease cleaves peptide chains mainly at the carboxyl side of lysine (K) and arginine (R) (Northrop and Kunitz 1931). However, trypsin activity on tagged lysines is greatly reduced due to the sterical hindrance of the TMT. One of the tryptic peptides generated from an intact Elp1p stretched from glutamic acid (E) at position 197 to arginine (R) at position 213 with a TMT-tagged K at position 203 (Figs. 3 and S1A).
In the same region, two tryptic peptides were generated from the truncated Elp1p, E197 -K203 with a TMT-tagged K at the peptide C-terminus (Figs. 3 and S1B), and A204 -R213 with a TMT on the peptide N-terminus (Figs. 3 and S1C). In order to verify the trypsin-digested sample, another batch of sample was digested using endopeptidase GluC in a buffered solution at pH 8.5 which makes the enzyme strongly favor cleavage after glutamic acid (E) over aspartic acid (D) (Birktoft and Breddam 1994). A GluC peptide generated from the intact Elp1p stretched from A198 -E233 with a TMT-tagged K at position 203 ( Fig. 3 and S1D), while two peptides generated from the truncated Elp1p were identified, an A198 -K203 with a TMT-tagged K at the peptide C-terminus (Fig. 3, spectra not shown), and an A204 -E233 with a TMT on the peptide N-terminus ( Fig. 3 and S1E). For both the trypsin-and GluC-digested samples, we could with high confidence identify a truncation site between position 203 and 204 (Figs. S1B, S1C, S1E and 1). Although we could find the C-terminal end of the truncated N-terminus of Elp1p (~26 kDa) in both samples, only the identification from the trypsin sample was unambiguous. In addition, the N-terminal of the larger   fragment (~140 kDa) was identified with high confidence in both samples. Thus, we conclude that the two major forms of Elp1p in the TAP-tag purification represent fulllength Elp1p and a shorter form of Elp1p cleaved between K203 and A204, hereafter called Elp1p Del N-term .
The Elp1p Del N-term is not active in wobble uridine tRNA modification To investigate if the Elp1p Del N-term is functional in wobble uridine tRNA modification, it was expressed in an elp1 null mutant. Both low and high copy plasmids were constructed where the elp1 gene encoding the Elp1p Del N-term (starting from A204) was cloned in frame with the AUG 2 translational start codon. In order to obtain a similar expression level as from the wild-type ELP1 gene, Elp1p Del N-term was expressed from a high copy vector (Fig. 4A). Total tRNA from the elp1 null mutant expressing the Elp1p Del N-term short form was isolated and analyzed for the presence of modified nucleosides. Expression of the Elp1p Del N-term did not complement the wobble uridine modification defect of an elp1 null mutant (Fig. 4B), indicating that Elp1p Del N-term is not active in tRNA modification.
Truncated Elp1p is a preparation artifact during sample preparation An intact Elongator complex was purified in the absence of Prb1p and formation of modified wobble uridines was not influenced in the prb1 null mutant ( Fig. 2A and C).
In addition to these observations, expression of the Elp1p Del N-term did not complement the tRNA modification defect As loading control, actin or tubulin were used.

Elp1p
Actin w t e l p 1 Δ p r b 1 Δ e l p 1 Δ + p r b 1 Δ w t e l p 1 Δ p r b 1 Δ of an elp1 null mutant strain (Fig. 4B). Therefore, we considered the possibility that the cleavage of Elp1p is an artifact taking place during sample preparation. To address this question, exponentially growing wild-type (BY4741), elp1Δ (UMY3906), and prb1Δ (YSC1053-YEL060C), strains were used to prepare cell pellets. One sample was also prepared where equal amounts of cell pellet from elp1Δ (UMY3906) and prb1Δ (YSC1053-YEL060C) cultures were mixed. Proteins from wild-type, elp1Δ, prb1Δ, and the mixed elp1Δ/ prb1Δ cell pellets were extracted. In the mixed elp1Δ/ prb1Δ cell pellet, Elp1p is only present in the prb1Δ strain and Prb1p is only present in the elp1Δ strain. Therefore, the appearance of truncated Elp1p would imply an in vitro endopeptidase cleavage by Prb1p originating from the elp1Δ strain occurring during sample preparation. Since a truncated Elp1p was detected from the mixed elp1Δ/ prb1Δ cell pellet (Fig. 5A, left panel), truncation of Elp1 occurred in vitro during sample preparation. To minimize a possible in vitro endopeptidase cleavage of Elp1p during sample preparation and to investigate whether truncated Elp1p could be observed in vivo in the wild-type strain (BY4741), a protein extraction method including TCA was used (Peter et al. 1993). In this method, the extracted proteins are precipitated with TCA immediately after cell lysis and denatured. In the previous study, where truncated Elp1p was observed, no TCA was included in the protein extraction (Fichtner et al. 2003). However, when TCA was included and Elp1p was detected by the anti-Elp1p antibody, an unspecific band occasionally appeared in the elp1Δ strain with almost the same size as truncated Elp1p (Fig. 5A, right panel). This inconsistency made it difficult to determine whether the Elp1p N-terminal truncation takes place in vivo or in vitro. To solve this problem, we made use of strains harboring Elp1-GFP protein fusions generating a slower migrating product. Thus, we used the ELP1-GFP (95700-YLR384C) strain and constructed an ELP1-GFP prb1Δ derivative (UMY3930). The experiment described above was repeated using strains ELP1-GFP (95700-YLR384C), ELP1-GFP prb1Δ (UMY3930), and elp1Δ (UMY3906). The Elp1-GFP protein was detected with an anti-GFP antibody, which did not give any unspecific signal at the position of truncated Elp1p. No truncated form of Elp1p was detected in pellets from wild-type or mixed elp1Δ/ prb1Δ cell pellets using the TCA method (Fig. 5B, right panel), whereas the truncated form was observed when TCA was excluded (Fig. 5B, left  panel). This result shows that the Elp1p N-terminal truncated form is generated during sample preparation. It was previously shown that more truncated Elp1p was observed in protein extracts from kti11Δ or urm1Δ mutants than from a wild-type strain (Fichtner et al. 2003). We considered that this observation might also be a result of a protein preparation artifact. To test this hypothesis, a western blot was performed to determine the relative amounts of full-length and truncated Elp1p in these mutants by extracting proteins in the absence or presence of TCA (Fig. 5C). When TCA was not included during protein extraction, more truncated Elp1p was observed in the kti11Δ and urm1Δ mutants compared to the wild-type strain (Fig. 5C,  left panel). This observation is similar as the previous finding (Fichtner et al. 2003). However, no truncated Elp1p was detected in the mutants when TCA was included (Fig. 5C,  right panel). These data support that appearance of truncated Elp1p is a preparation artifact. The reason why more truncated Elp1p was observed when TCA was excluded during protein extraction might be that Kti11p and Urm1p protect Elp1p from cleavage of Prb1p.
Apparently, in the presence of Prb1p without using TCA during protein extraction, truncation of Elp1p is prone to occur. Prb1p is a vacuolar protease and the vacuole serves as a compartment to degrade cytoplasmic proteins and organelles by autophagy under nitrogen starvation (Li and Kane 2009). Therefore, it will not be surprising that under nitrogen starvation Elp1p is delivered to vacuole, trimmed by Prb1p, and degraded.
Although both protein extraction buffers contain a protease inhibitor cocktail, it is obviously not enough to inhibit the action of protease Prb1p. Thus, to be sure to circumvent cleavage or degradation of proteins to be studied, alternative protein extraction methods should be used to avoid misinterpretations of the data.