Kex1 Protease Is Involved in Yeast Cell Death Induced by Defective N-Glycosylation, Acetic Acid, and Chronological Aging
2008; Elsevier BV; Volume: 283; Issue: 27 Linguagem: Inglês
10.1074/jbc.m801303200
ISSN1083-351X
AutoresPeter Hauptmann, Ludwig Lehle,
Tópico(s)Redox biology and oxidative stress
ResumoN-Glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast. N-Glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to humans. The key step of this pathway is the transfer of the lipid-linked core oligosaccharide to the nascent polypeptide chain, catalyzed by the oligosaccharyltransferase complex. Temperature-sensitive oligosaccharyltransferase mutants of Saccharomyces cerevisiae at the restrictive temperature, such as wbp1-1, as well as wild-type cells in the presence of the N-glycosylation inhibitor tunicamycin display typical apoptotic phenotypes like nuclear condensation, DNA fragmentation, phosphatidylserine translocation, caspase-like activity, and reactive oxygen species accumulation. Since deletion of the yeast metacaspase YCA1 did not abrogate this death pathway, we postulated a different proteolytic process to be responsible. Here, we show that Kex1 protease is involved in the programmed cell death caused by defective N-glycosylation. Its disruption decreases caspase-like activity, production of reactive oxygen species, and fragmentation of mitochondria and, conversely, improves growth and survival of cells. Moreover, we demonstrate that Kex1 contributes also to the active cell death program induced by acetic acid stress or during chronological aging, suggesting that Kex1 plays a more general role in cellular suicide of yeast. Apoptosis is a highly regulated suicide program crucial for the development and maintenance of higher eukaryotes, and its dysfunction is the cause of several diseases (1Kerr J.F. Toxicology. 2002; 181: 471-474Crossref PubMed Scopus (205) Google Scholar, 2Aridor M. Balch W.E. Nat. Med. 1999; 5: 745-751Crossref PubMed Scopus (255) Google Scholar, 3Paschen W. Frandsen A. J. Neurochem. 2001; 79: 719-725Crossref PubMed Scopus (209) Google Scholar, 4Kouroku Y. Fujita E. Jimbo A. Kikuchi T. Yamagata T. Momoi M.Y. Kominami E. Kuida K. Sakamaki K. Yonehara S. Momoi T. Hum. Mol. Genet. 2002; 11: 1505-1515Crossref PubMed Scopus (168) Google Scholar, 5Hetz C. Russelakis-Carneiro M. Maundrell K. Castilla J. Soto C. 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Kollroser M. Frohlich K.U. Sigrist S. Madeo F. Mol. Cell. 2007; 25: 233-246Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), and a caspase-like protein (Yca1/Mca1p) belonging to a new family of caspases, the so-called metacaspases (33Madeo F. Herker E. Maldener C. Wissing S. Lachelt S. Herlan M. Fehr M. Lauber K. Sigrist S.J. Wesselborg S. Frohlich K.U. Mol. Cell. 2002; 9: 911-917Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar).In addition, physiological roles for yeast apoptosis are emerging during chronological aging, by providing a better regrowth of fitter, better-adapted cells (34Herker E. Jungwirth H. Lehmann K.A. Maldener C. Frohlich K.U. Wissing S. Buttner S. Fehr M. Sigrist S. Madeo F. J. Cell Biol. 2004; 164: 501-507Crossref PubMed Scopus (448) Google Scholar) promoted through the release of nutrients (35Fabrizio P. Battistella L. Vardavas R. Gattazzo C. Liou L.L. Diaspro A. Dossen J.W. Gralla E.B. Longo V.D. J. Cell Biol. 2004; 166: 1055-1067Crossref PubMed Scopus (299) Google Scholar). Similarly, regulated cell death has been shown to be essential during yeast development for the long term survival of the colony population in order to adapt to the environment, whereby ammonia acts as a signal for differentiation (36Vachova L. Palkova Z. J. Cell Biol. 2005; 169: 711-717Crossref PubMed Scopus (152) Google Scholar, 37Vachova L. Devaux F. Kucerova H. Ricicova M. Jacq C. Palkova Z. J. Biol. Chem. 2004; 279: 37973-37981Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).We have recently shown that a defect in N-glycosylation exhibits typical apoptotic cellular phenotypes. N-Glycosylation is one of the most common types of eukaryotic protein modifications, and the pathway is highly conserved from yeast to humans (38Lehle L. Strahl S. Tanner W. Angew Chem. Int. Ed Engl. 2006; 45: 6802-6818Crossref PubMed Scopus (238) Google Scholar). In humans, defects in N-glycosylation are the cause of congenital disorders of glycosylation, a new family of genetic diseases with a severe, multisystemic clinical picture (39Jaeken J. Carchon H. Curr. Opin. Pediatr. 2004; 16: 434-439Crossref PubMed Scopus (85) Google Scholar). We demonstrated that yeast mutants with a defect in subunits of the oligosaccharyltransferase (OST), 2The abbreviations used are: OST, oligosaccharyltransferase; AMC, 7-amino-4-methylcoumarin; DHR, dihydrorhodamine; DHE, dihydroethidium; D2R, aspartate2 rhodamine 110; FITC, fluorescein isothiocyanate; fmk, fluoro-methylketone; GFP, green fluorescent protein; H2DCFDA, 2′,7′dichloro dihydrofluorofluorescein diacetate; PI, propidium iodide; ROS, reactive oxygen species; TM, tunicamycin; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; mtGFP, mitochondrion-targeted green fluorescent protein construct. 2The abbreviations used are: OST, oligosaccharyltransferase; AMC, 7-amino-4-methylcoumarin; DHR, dihydrorhodamine; DHE, dihydroethidium; D2R, aspartate2 rhodamine 110; FITC, fluorescein isothiocyanate; fmk, fluoro-methylketone; GFP, green fluorescent protein; H2DCFDA, 2′,7′dichloro dihydrofluorofluorescein diacetate; PI, propidium iodide; ROS, reactive oxygen species; TM, tunicamycin; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; mtGFP, mitochondrion-targeted green fluorescent protein construct. such as wbp1 and ost2, display morphological and biochemical features of apoptosis. OST2 from yeast is homologous to DAD1 (40Knauer R. Lehle L. Biochim. Biophys. Acta. 1999; 1426: 259-273Crossref PubMed Scopus (170) Google Scholar), originally characterized in hamster cells as a "defender against apoptotic death" (41Nakashima T. Sekiguchi T. Kuraoka A. Fukushima K. Shibata Y. Komiyama S. Nishimoto T. Mol. Cell Biol. 1993; 13: 6367-6374Crossref PubMed Scopus (205) Google Scholar, 42Niederer K.E. Morrow D.K. Gettings J.L. Irick M. Krawiecki A. Brewster J.L. Cell. Signal. 2005; 17: 177-186Crossref PubMed Scopus (25) Google Scholar). We observed nuclear condensation, DNA fragmentation, and externalization of phosphatidylserine. We also provided evidence for the production of reactive oxygen species (ROS) and a caspase-like activity, which could be diminished by heterologous expression of antiapoptotic human Bcl-2. Since deletion of the yeast metacaspase YCA1/MCA1 did not seem to abrogate this activity, we postulated a different proteolytic activity to be involved in cell death induced by a protein N-glycosylation defect (24Hauptmann P. Riel C. Kunz-Schughart L.A. Frohlich K.U. Madeo F. Lehle L. Mol. Microbiol. 2006; 59: 765-778Crossref PubMed Scopus (95) Google Scholar).Searching for a potential protease involved in this process, we came across Kex1. This protein has been characterized as a serine carboxypeptidase B-like protease, specific for basic amino acid residues, responsible for processing of prepro-α-factor (mating pheromone) as well as K1 and K2 killer toxin precursors (43Bussey H. Yeast. 1988; 4: 17-26Crossref PubMed Scopus (61) Google Scholar, 44Fuller R.S. Sterne R.E. Thorner J. Annu. Rev. Physiol. 1988; 50: 345-362Crossref PubMed Scopus (337) Google Scholar) while traversing the secretory pathway. Kex1 localizes to the Golgi apparatus and contains a membrane-spanning domain at the carboxyl-terminal side, whereas the large protease domain at the NH2 terminus is extended into the lumen of the secretory pathway (45Cooper A. Bussey H. J. Cell Biol. 1992; 119: 1459-1468Crossref PubMed Scopus (68) Google Scholar).In this study, we demonstrate that Kex1 protease is a new player in the yeast cell death cascade caused by defective N-glycosylation both in the glycosylation mutant wbp1-1 and in wild-type cells when exposed to the N-glycosylation inhibitor tunicamycin. Disruption of KEX1 diminishes caspase-like activity and ROS accumulation, retards the fragmentation of mitochondria, and leads to a better growth and survival of cells. Furthermore, we provide evidence that KEX1 is also involved in cell death provoked by acetic acid stress and during chronological aging.EXPERIMENTAL PROCEDURESYeast Strains, Media, and Genetic MethodsThe following strains were used: SS328 (MATα ade2-101 ura3-52 his3Δ200 lys2-801), SS328Δkex1 (MATα ade2-101 ura3-52 his3Δ200 lys2-801 Δkex1::kanMX), SS328Δyca1 (MATα ade2-101 ura3-52 his3Δ200 lys2-801 Δyca1:: kanMX), WCA (MATα his3-11,15 leu2-3,112 ura3), WCAΔpep4 (MATα his3-11,15 leu2-3,112 ura3 Δpep4::HIS3), WCAΔpep4Δkex1 (MATα his3-11,15 leu2-3,112 ura3 Δpep4::HIS3 Δkex1::kanMX), MA7-B (MATa ade2-101 ura3-52 his3Δ200 lys2-801 wbp1-1), MA7-BΔkex1 (MATa ade2-101 ura3-52 his3Δ200 lys2-801 wbp1-1 Δkex1::kanMX), MA7-BΔyca1 (MATa ade2-101 ura3-52 his3Δ200 lys2-801 wbp1-1 Δyca1::kanMX), MA7-BΔaif1 (MATa ade2-101 ura3-52 his3Δ200 lys2-801 wbp1-1 Δaif1::kanMX), MA7-BΔnma111 (MATa ade2-101 ura3-52 his3Δ200 lys2-801 wbp1-1 Δnma111::kanMX). Strains were grown in YPD (1% yeast extract, 2% bacto-peptone, 2% glucose) or in selective YNB (yeast nitrogen base) medium supplemented with amino acids, nucleotide bases, as required, and 2% glucose at the temperatures indicated. For osmotic stabilization, 1 m sorbitol was added to rich medium.Disruption of KEX1, YCA1, AIF1, and NMA111 was carried out with a kanMX cassette amplified by PCR from the plasmid pUG6 (46Guldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1341) Google Scholar), using 5′-GCATACTTTGGTTAAAGAGTACCTTGGCTATAGAATACCGTAGAGATAAAGACCcagctgaagcttcgtacgc-3′ and 5′-GCCAAGTTAAAAATCAGTCATCTCAAAAGATTCATCGATTTCAGTGTTCGGAAGGCgcatagcccactagtggatct-3′ for disruption of KEX1 and using 5′-GCGTCCGGGTAATAACAACTATTGAAAAAGCATGGCTTCGCATTAATAGGAGCCcagctgaagcttcgtacgc-3′ and 5′-TACATAATAAATTGCAGATTTACGTCAATAGGGTGTGACGATGATAATTGTGGgcatagcccactagtggatct-3′ for disruption of YCA1. To delete AIF1 and NMA111, the kanMX cassettes were amplified by PCR using 5′fw primer 5′-GCAGTGATAAATCATTGATAGACACACATTTCCTCATCGTTGTTCTTCATTATTTACAGGAAAGAGCcagctgaagcttcgtacgc-3′ and 3′rev primer 5′-GCTGCAGTTCATATTTTAGTCTATTTTTTGAATAAAGGTTCCATTTTGTCGGAGAAAAGATTCTTCGgcatagcccactagtggatct for AIF1 and using 5′-GTAGAGTACAGTAAAGGTTTTTTAGATCTACTAATGACCATATCGTTGAGCcagctgaagcttcgtacgc-3′ and 5′-CTATTTTTCACTTTGGCTGTTGCCGGTAAATTCC TTTTCAATCCATTTGTGCGgcatagcccactagtggatct-3′ for NMA111. The cassettes were transformed into yeast strains SS328 (wild-type) and MA7-B (wbp1-1). Recombinants were selected for resistance to G418 sulfate, and positive clones were verified by PCR.To construct the expression plasmid pVT100-KEX1-ZZ, KEX1 was amplified by PCR with genomic DNA from wild-type strain SS328 as template using primer 5′-CCGAAGCTTATGTTTTACAATAGG-3′ with a HindIII site and primer 5′-TGCTCTAGAAAAATCAGTCATCTC-3′ with an XbaI site. The insert was ligated in HindIII/XbaI-cut yeast shuttle vector pVT100-ZZ. Correctness of the construct was verified by sequencing and analysis of the protein by Western blotting using the protein A epitopes (ZZ) fused in frame to the COOH terminus for detection.For tagging genomic KEX1 with two ZZ-epitopes, a cassette was amplified by PCR with 5′-GCCTTCCGAACACTGAAATCGATGAATCTTTTGAGATGACTGATTTGGAGCAGGGGCGGGTGC-3′ and 5′-CCCTTTAAAGAATTTATCTTTATGTCGCTGTTACTACGAAAAGCGTGTGCGAGGTCGACGGTATCG-3′ as primers and using plasmid pZZ-KAN as template. Transformants were selected on G418-sulfate medium, and the correct recombination at the chromosomal KEX1 locus was verified by PCR using the primers 5′-GTTACGCTGGCCAATACATACC-3′ and 5′-GAATTTTCGTTTTAAAACCTAAGAGTCAC-3′ and expression of the tagged protein by Western analysis. Mitochondria were visualized using a mitochondria-targeted green fluorescent protein in strains transformed with plasmid pVT100-mtGFP, as described previously (47Westermann B. Neupert W. Yeast. 2000; 16: 1421-1427Crossref PubMed Scopus (309) Google Scholar).Induction of Programmed Cell Death by Temperature Shift and Treatment with Acetic AcidFresh cultures were grown to a cell density of 2 × 106 ml-1 overnight at 25 °C and divided into two flasks. For initiation of the cell death program in wbp1-1, cultures were shifted to restrictive temperatures as indicated. For treatment with acetic acid, cells were harvested at a cell density of 2 × 106 ml-1 and suspended in YPD medium (pH 3.0; adjusted with HCl) containing appropriate concentrations of acetic acid at a cell density of 3 × 106 ml-1. Treatment with acetic acid was carried out for 200 min at 25 °C (16Ludovico P. Sousa M.J. Silva M.T. Leao C. Corte-Real M. Microbiology. 2001; 147: 2409-2415Crossref PubMed Scopus (408) Google Scholar).Confocal Laser Microscopy and Flow CytometryFor detection of caspase-like activity, 5 × 106 cells were harvested, washed in phosphate-buffered saline, and resuspended in 0.2 ml of staining solution containing 10 μm FITC-VAD-fmk in phosphate-buffered saline (CaspACE FITC-VAD-fmk in situ marker; Promega) or in 0.25 ml of YPD containing 0.2 mm D2R (25 mm stock solution in ethanol/dimethyl sulfoxide 2:1; Thermo Scientific). After incubation for 20 min at room temperature (for FITC-VAD-fmk) or 30 min at 30 °C (for D2R) with low agitation in darkness, cells were centrifuged and washed twice with 1 ml of phosphate-buffered saline. Free intracellular radicals (ROS) were detected by dihydrorhodamine 123 (DHR123) and 2′,7′-dichlorodihydro-fluoresceindiacetate (H2DCFDA), which were added 1.5-2 h before harvesting the cells from a 2.5 mg ml-1 stock solution in ethanol to a final concentration of 5 μg ml-1. Staining with dihydroethidium (DHE) was performed as described in Ref. 32Buttner S. Eisenberg T. Carmona-Gutierrez D. Ruli D. Knauer H. Ruckenstuhl C. Sigrist C. Wissing S. Kollroser M. Frohlich K.U. Sigrist S. Madeo F. Mol. Cell. 2007; 25: 233-246Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar. For staining with propidium iodide (PI), cells were treated as described for caspase staining. PI was added from a 50 μg ml-1 stock solution in water to a final concentration of 50 ng ml-1.For confocal laser microscopy, cells were concentrated by a short centrifugation step and immobilized by covering with 1% agarose. Analysis was performed using an LSM 510-META confocal laser-scanning microscope (Carl Zeiss, Jena, Germany). For detection of FITC-VAD-fmk, D2R, DHR123, H2DCFDA, and GFP fluorescence signals, a 505-550-nm band pass emission filter was used with an excitation at 488 nm (argon laser). PI fluorescence (excitation 543 nm, HeNe laser) was ascertained using a 585-nm long pass emission filter. To avoid cross-talk between the fluorescence channels in the case of co-staining, probes were scanned sequentially.For flow cytometric analysis, cells were resuspended in 500 μl of phosphate-buffered saline, and 20,000 cells were probed. Analysis was achieved using the MoFlo (Cytomation) high speed sorter and Summit version 3.1 software. FITC-VAD-fmk, D2R, DHR123, and H2DCFDA fluorescence signals were determined using a 510-550-nm band pass filter; for PI and DHE, a 610-650-nm band pass filter was used (excitation 488 nm, argon laser).Test for Determination of Cell SurvivalTo determine the survival rate, cells were separated by the MoFlo cell sorter and spotted onto YPD plates. For each measuring point, 250 single cells were plated, and the number of colonies was determined after 3-4 days of incubation at the indicated temperature.Assay for KEX1 ActivityMembrane Preparation—Yeast cells were grown in minimal medium (0.67% yeast nitrogen base, 0.5% casamino acids, 20 mg ml-1 tryptophan, 20 mg ml-1 adenine, 30 mg ml-1 tyrosine, 2% glucose) overnight to a cell density of 2 × 107 ml-1. Approximately 3 × 1010 cells were harvested at 4,000 × g for 10 min at 4 °C, washed with 20 ml of 50 mm Tris-HCl (pH 7.5), and resuspended in buffer A containing 50 mm Tris-HCl (pH 7.5), 1 mm EtSH, 1 mm benzamidine, 1 mm MgCl2 and 5% glycerol; per gram, wet weight, 1 ml of buffer was added. Cells were broken with glass beads using a Merckenschlager bead beater (Braun, Melsungen, Germany), and the cell lysate was filtered to remove beads. After centrifugation at 500 × g for 5 min at 4 °C, the supernatant was centrifuged at 48,000 × g for 30 min at 4 °C. The pellet was washed once and resuspended in buffer B containing 30 mm Tris-HCl (pH 7.5), 3 mm MgCl2, 1 mm dithiothreitol, and 35% glycerol.Purification of KEX1-ZZ—For solubilization of KEX1-ZZ from the membrane fraction, 1.2 mg of protein in buffer B containing 0.5 m KCl and 1% octyl glucoside (added dropwise) were incubated in a final volume of 0.2 ml for 20 min on ice. The mixture was centrifuged at 150,000 × g for 40 min at 2 °C, and 0.1 ml of the supernatant was added to 50 μl of IgG-Sepharose equilibrated with 50 mm Tris-HCl (pH 7.5) and 150 mm NaCl. After incubation by rolling end over end for 30 min at 4 °C, the Sepharose beads were centrifuged at 300 × g for 3 min at 4 °C and washed three times with 25 mm BisTris-HCl (pH 6.3).Enzyme Tests—To determine Kex1-ZZ activity, 0.5 mm furylacryloyl-Ala-Arg-OH in methanol and 930 μl of 25 mm Bis-Tris-HCl (pH 6.3) were added to 50 μl of IgG-Sepharose beads to which Kex1-ZZ was adsorbed as prepared above and incubated for the times indicated. Beads were centrifuged at 13,000 × g for 15 s, and from the supernatant, the absorption at 340 nm was determined. To investigate a caspase-like activity, 40 μm Ac-VEID-AMC (2 mm stock solution in DMSO) was added to Kex1-ZZ-adsorbed IgG-Sepharose beads as described before. After incubation for 2 h by rolling end over end, emission of the supernatant was determined at a wavelength of 440 nm, applying an excitation of 370 nm. Measurements were accomplished in a Jobin Yvon-Spec Fluoromax-2 spectrofluorimeter with DataMax software.In Vivo Labeling with [35S]Methionine/CysteineYeast cells were grown in YPD to a cell density of 7 × 106 ml-1 overnight at 25 °C and then shifted to 37 °C for the times indicated. Prior to labeling, cells were transferred for 30 min to YNB glucose medium, and subsequently cells were labeled for 30 min at 37 °C and further processed for isolation and PAGE analysis of carboxypeptidase CPY as described in Ref. 48Knauer R. Lehle L. J. Biol. Chem. 1999; 274: 17249-17256Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar.RESULTSDisruption of KEX1 Causes Better Growth and Survival of N-Glycosylation-defective Cells and Decreases Caspase-like Activity as Well as ROS Accumulation—Wbp1 is an essential subunit of the oligosaccharyltransferase complex, the key enzyme of protein N-glycosylation (49Knauer R. Lehle L. FEBS Lett. 1994; 344: 83-86Crossref PubMed Scopus (59) Google Scholar, 50te Heesen S. Janetzky B. Lehle L. Aebi M. EMBO J. 1992; 11: 2071-2075Crossref PubMed Scopus (118) Google Scholar). When the temperature-sensitive mutant wbp1-1 is shifted from the permissive temperature of 25 °C to a restrictive temperature, an underglycosylation of glycoproteins occurs, and cells start to die, thereby exhibiting typical markers of apoptosis (24Hauptmann P. Riel C. Kunz-Schughart L.A. Frohlich K.U. Madeo F. Lehle L. Mol. Microbiol. 2006; 59: 765-778Crossref PubMed Scopus (95) Google Scholar). As shown in Fig. 1A, disruption of KEX1 restores the temperature growth defect of wbp1-1 at 31 °C. Similarly, a better survival of cells is observed. For this purpose, cells were shifted for different time periods to the restrictive temperature of 37 °C and subsequently separated with a cell sorter, and single cells were spotted on full medium plates. After 4 days at 25 °C, survival of cells was determined (Fig. 1B). The positive effect on survival by deletion of KEX1 can also be observed at restrictive temperatures lower than 37 °C (see supplemental Fig. 1). Deletion of KEX1 in wild-type cells has no effect on growth (Fig. 1A) or survival (data not shown), indicating that the N-glycosylation defect in wbp1 is the cause. A terminal, visible phenotype occurring in yeast programmed cell death is an alteration of the cell morphology, characterized by cell shrinkage. As shown in Fig. 1C, in wbp1-1Δkex1 cells lacking Kex1 protease, the amount of shrunken cells was strongly reduced from 20 to 5%, in comparison with wbp1-1 cells upon a shift to 37 °C for 4 h.We have recently demonstrated a caspase-like, proteolytic activity both by cell flow cytometry and cell-free extracts of N-glycosylation-stressed cells using typical metazoan caspase substrates (24Hauptmann P. Riel C. Kunz-Schughart L.A. Frohlich K.U. Madeo F. Lehle L. Mol. 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Cell Sci. 2002; 115: 4727-4734Crossref PubMed Scopus (425) Google Scholar, 54Borner C. Monney L. Cell Death Differ. 1999; 6: 497-507Crossref PubMed Scopus (232) Google Scholar). Therefore, we asked whether Kex1 contributes to the proteolytic activity measured during cell death of wbp1-1 and how it compares with Yca1. As shown in Fig. 2A, deletion of Kex1 protease in wbp1-1 leads to a strong decrease of the amount of cells labeled with cell-permeable FITC-valyl-alanylaspartyl-(O-methyl)-fluoromethylketone (FITC-VAD-fmk), when analyzed by cell flow cytometry. Although 32 and 64%, respectively, of wbp1-1 cells showed positive staining after a temperature shift to 37 °C for 4 and 8 h, this amounted to only 10 and 25% in wbp1-1Δkex1 cells. In wild-type cells and wild-type Δkex1, no staining at 37 °C was detectable. The positive influence by KEX1 occurs already at lower restrictive temperatures than 37 °C (supplemental Fig. 1B). In contrast to KEX1, disruption of YCA1 in wbp1-1 had no effect on the caspase-like activity under the same conditions (Fig. 2A). In cells cultured at the permissive temperature of 25 °C, hardly any activity could be measured. Similarly, disruption of YCA1 affects neither growth (Fig. 1A), survival (Fig. 1B), nor cell shr
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