Kex1 Protease Is Involved in Yeast Cell Death Induced by Defective N-Glycosylation, Acetic Acid, and Chronological Aging

Abstract

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 (1-7). For a long time, the opinion prevailed that apoptosis is a process characteristic only of multicellular organisms, but recent studies convincingly demonstrate that forms of programmed cell death with apoptotic-like phenotypes are also present in unicellular organisms such as bakers' yeast (6, 8 -13).After the first discovery of apoptotic phenotypes in a cdc48 mutant (14), it was found that yeast undergoes apoptotic-like death after application of hydrogen peroxide (15) In addition, physiological roles for yeast apoptosis are emerging during chronological aging, by providing a better regrowth of fitter, better-adapted cells (34) promoted through the release of nutrients (35). 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 (36,37).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 (38). 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 (39). We demonstrated that yeast mutants with a defect in subunits of the oligosaccharyltransferase (OST), 2 such as wbp1 and ost2, display morphological and biochemical featur...

Nonapoptotic Death of Saccharomyces cerevisiae Cells That Is Stimulated by Hsp90 and Inhibited by Calcineurin and Cmk2 in Response to Endoplasmic Reticulum Stresses

“…Therefore, little support exists for the ability of tunicamycin to induce apoptosis in wild-type S. cerevisiae in YPD medium. Recently, this manner of cell death was shown to depend on Kex1, a Golgi-localized carboxypeptidase that is also important in the death of mutant cells lacking certain N-glycosylation factors and in the death of yeast cells in response to other stresses (26). The addition of an osmotic stabilizer to the culture medium also blocked death in these conditions, suggesting that the manner of cell death may be lysis.…”

“…A previous study arrived at the opposite conclusion (27), but the analysis was flawed by the failure to distinguish live apoptotic cells from dead cells, which are known to stain nonspecifically with FITC-VAD-FMK and other fluorophores, such as DHR123 (66). Very recently, the yeast metacaspase (Mca1) was shown to be unimportant for tunicamycin-induced cell death in YPD medium (26). Therefore, little support exists for the ability of tunicamycin to induce apoptosis in wild-type S. cerevisiae in YPD medium.…”

Calnexin Is Involved in Apoptosis Induced by Endoplasmic Reticulum Stress in the Fission Yeast

“…Tunicamycin is an inhibitor of N-glycosylation that strongly induces apoptosis in mammalian cells and in the budding yeast (Perez-Sala and Mollinedo, 1995; Hacki et al, 2000;Hauptmann et al, 2006;Hauptmann and Lehle, 2008). We have previously reported that tunicamycin efficiently inhibits protein N-glycosylation and induces ER stress in S. pombe (Jannatipour and Rokeach, 1995;Jannatipour et al, 1998;Beaulieu et al, 1999).…”

“…An increase in the calnexin levels due to ER stress may constitute a part of a branch in the mechanism of induction of apoptotic death. Tunicamycin is a potent inhibitor of N-glycosylation that is currently used as an elicitor of ER stress in the study of the apoptosis mechanisms in mammalian cells and in the budding yeast (Perez-Sala and Mollinedo, 1995; Hacki et al, 2000;Hauptmann et al, 2006;Hauptmann and Lehle, 2008). We have previously reported that tunicamycin induces ER stress in S. pombe and increases the expression of cnx1 ϩ , the gene encoding calnexin (Jannatipour and Rokeach, 1995).…”

Serine-Type Carboxypeptidase KexA of Aspergillus oryzae Has Broader Substrate Specificity than Saccharomyces cerevisiae Kex1 and Is Required for Normal Hyphal Growth and Conidiation

“…Kex1 is involved in the proteolytic processing of ␣-factor and of S. cerevisiae killer toxin (3,4,5,8,12,17). In addition, recent studies have shown that Kex1 plays a more general role in yeast (9,10). A whole-genome BLAST search revealed that the KEX1 orthologous gene is also present in the genome of the Aspergillus species; however, there are no reports on the enzymatic or functional properties of the Kex1 ortholog in the filamentous fungi.…”