is a form of programmed cell death with an important role in development and homeostasis of metazoan organisms. Apoptosis allows the rapid removal of unwanted or damaged cells that could otherwise inflame the surrounding cells with their cytoplasmic contents. In contrast, during necrosis, a form of cell death that results from overwhelming cellular injury, cells lyse and release cytoplasmic material. The apoptotic program is switched on in irreparably damaged or potentially dangerous cells such as self-reactive lymphocytes or cells that have been infected by viruses. Furthermore, it is involved in tumor suppression and in a wide range of diseases such as AIDS, neurodegenerative processes, and ischemic stroke (Steller, 1995
Apoptotic cells are characterized by a set of distinct morphological changes (Kerr et al., 1972
; Wyllie, 1980
; Wyllie et al., 1980
). An early marker of apoptosis is the exposition of phosphatidylserine on the cell surface, whereas it is normally concentrated in the luminal layer of the cytoplasmic membrane (Martin et al., 1995
). DNA is cleaved between nucleosomes (Wyllie, 1980
) and the chromatin condenses (Kerr et al., 1972
), typically starting as a ring at the inner side of the nuclear envelope (Clifford et al., 1996
). Finally, cells break up into membrane-enclosed fragments, the apoptotic bodies (Kerr et al., 1972
), which are rapidly phagocytosed and digested by macrophages.
The initiation of apoptosis is a highly coordinated and regulated process. It can be induced or suppressed by a lot of different intracellular and extracellular signals such as Bcl-2 family proteins (Bax, Bak, and Bcl-2), IL-1β–converting enzyme (ICE)1
proteases (caspases), and p53. p53 is the most frequently altered gene in human cancers. DNA damage induces p53 expression, which leads to cell cycle arrest at G1
or to induction of apoptosis (Donehower and Bradley, 1993
). Bax induces apoptosis in mammalian cells by the activation of ICE proteases (Chinnaiyan et al., 1996
), which mediate the cleavage of several proteins including those of the nuclear matrix and nuclear envelope, finally leading to DNA fragmentation.
Though no proteins homologous to any of these apoptotic regulators have been detected in the yeasts Schizosaccharomyces pombe
and Saccharomyces cerevisiae
, the expression of Bax or p53 causes a severe effect on cell proliferation. Overexpression of human p53 inhibits cell growth of S. pombe
and S. cerevisiae
as in higher eukaryotes, however it does not result in an apoptotic phenotype (Bischoff et al., 1992
). Expression of Bax in the yeast S. pombe
is lethal, coexpression of the anti-apoptotic protein Bcl-2 suppresses this effect. As in mammals, Bcl-2 appears to inactivate Bax by forming mixed dimers: a mutant of Bcl-2 that fails to heterodimerize with Bax does not rescue yeast cell growth (Jürgensmeier et al., 1997
). However, Bax-induced cell death in S. pombe
is not accompanied by any classical morphological feature of apoptosis. Neither evidence of nuclear fragmentation nor of chromatin margination against the nuclear envelope, nor internucleosomal DNA fragmentation was observed. Jürgensmeier et al. (1997)
, therefore labeled the Bax-induced effect “cytotoxicity,” to distinguish it from apoptosis.
We investigated a mutant of baker's yeast in cell division cycle gene CDC48
for markers of apoptosis. Cdc48p plays an important role in the homotypic fusion of the endoplasmic reticulum; in vitro, Cdc48p is the only soluble protein necessary for the fusion of ER-derived vesicles (Latterich et al., 1995
). A defect in CDC48
results in a cell cycle arrest as a large budded cell with the nucleus located in the neck between the mother and the daughter cell at 16°C (Moir et al., 1982
; Fröhlich et al., 1991
Cdc48p is a member of the AAA family, which is characterized by a highly conserved element of ~230 amino acid residues, containing an ATP binding consensus sequence that can be present singly or in two copies. A database of the AAA family is available on the World Wide Web at http://yeamob.pci.chemie.uni-tuebingen.de/
We describe a cdc48 mutant of baker's yeast displaying several of the characteristic morphological markers of apoptosis. This is the first indication that the basic machinery of apoptosis is already present in unicellular lower eukaryotes.