Laboratory of Cell Division Cycle, Intracellular Transport and Apoptosis
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CDC48p and its Homologues

Characterization of CDC48p

A conditional S. cerevisiae CDC48 mutant arrests with a large bud at non-permissive conditions. In the arrested cells, abnormal spindle fibers spread from an unseparated spindle pole body. Isolated CDC48p forms annular homohexamers and exhibits an NEM inhibitable ATPase activity.
CDC48p homologues have been found in vertebrates (VCP in pig and mouse, TER-ATPase in rat, p97 in Xenopus laevis), plants, and archebacteria (SAV in Sulfolobus acidocaldarius, cdcH in Halobacterium salinarium). CDC48p and its vertebrate homologues exhibit a sequence identity of 69%, an unusually high value for the evolutionary distance (for comparison: the sequence identity between yeast Sec18p and its mammalian homologue NSF is 45%). The degree of sequence identity between CDC48p (or VCP) and SAV, 43%, make them one of the best conserved sequence pairs between Archaea and Eukaryotes. CDC48p and VCP have an essential role in the homotypic fusion of ER respectively Golgi vesicles.

Functionality of segments of the Cdc48p orthologue VCP in Saccharomyces cerevisiae

Though Cdc48p and porcine VCP show a high sequence conservation - almost 70% of their amino acid residues are identical - the VCP gene fails to complement a disruption of CDC48. Complementation studies with CDC48 and VCP gene hybrids show that an exchange of the central Cdc48p domain for the central VCP domain prevents a complementation of a CDC48 disruption, although this is the best conserved region between the two proteins. Protein chimeras containing the amino terminal part of VCP only complement a disruption of CDC48 when expressed at high levels. The respective yeast strain shows a nucleus devoid of Cdc48p. In contrast to VCP, Cdc48p contains an almost perfect nuclear targeting sequence in this region. Exchange of the carboxy terminal Cdc48p domain for the carboxy terminus of VCP leads to normal viability of the cell even at low expression levels.

Regulation of the intracellular localization of CDC48p and its human homologue

Both Cdc48p and its highly conserved mammalian homologue VCP are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle dependent manner: Cdc48p enters the nucleus during late G1, VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle dependent phosphorylation of a tyrosine residue near the carboxy terminus. When the residue is changed to a phenylalanine residue (imitating a non-phosphorylated tyrosine) by site-directed mutagenesis, the protein is excluded from the nucleus, and cell growth is slowed. If the residue is changed to a glutamic acid (mimicking a phosphotyrosine), the mutant protein is concentrated in the nucleus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein exposing the nuclear import signal sequence previously masked by a stretch of acidic residues. A deletion of this acidic region partially restores nuclear import of the phenylalanine mutant protein.

The AAA Protein Superfamily Database

CDC48p belongs a protein superfamily whose members have been detected in all phylogenetic kingdoms (Archaea, Eubacteria, Eukaryota) and which are involved in a number of crucial cellular activities. The proteins of the superfamily share a conserved region of app. 230 amino acid residues which can be present either as a single copy or as a tandem duplicate. The "conserved region" contains an ATP binding consensus sequence. Because of these characteristics, the name "AAA Superfamily" (ATPases Associated with diverse cellular Activities) has been proposed (Kunau et al., 1993).
We have assembled information about all known members (currently >200) of the AAA superfamily, which are updated regularly and which are available to the scientific community as the AAA Database on the WWW.

A yeast mutant shows diagnostic markers of early and late apoptosis

Apoptosis is a form of programmed cell death with an important role in development and homeostasis of metazoan organisms. Apoptosis allows the rapid removing of unwanted or damaged cells that otherwise could inflammate 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 programm 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.
Apoptotic cells are characterized by a set of distinct morphological changes. An early marker of apoptosis is the exposition of phosphatidylserine on the cell surface, while normally it is concentrated in the luminal layer of the cytoplasmic membrane. DNA is cleaved between nucleosomes and the chromatin condenses, typically starting as a ring at the inner side of the nuclear envelope. Finally, cells break up into membrane-enclosed fragments, the apoptotic bodies, that are rapidly phagocytosed and digested by macrophages.

We found that a Saccharomyces cerevisiae mutant in cell division cycle gene CDC48 (cdc48S565G) shows typical markers of apoptosis: membrane staining with annexin V indicating an exposure of phosphatidylserine, intense TUNEL staining indicating DNA fragmentation, and chromatin condensation and fragmentation. The coordinate occurrence of these events at different locations in the cell which have no obvious connection except their relation to apoptosis implies the presence of the molecular machinery performing the basic steps of apoptosis already in yeast :-).
We found that apoptosis can be induced in Saccharomyces cerevisiae by oxygen radicals provoked either by depletion of glutathione in a gsh1 deletion strain, or by treating cells with low doses of H2O2. Yeast apoptosis caused by cdc48S565G or due to the expression of a mammalian bax gene is also accompanied by an accumulation of oxygen radicals. In mammalian apoptosis, oxygen radicals serve as inducers both in early and late stages of the process. Our results indicate a functional conservation of part of apoptotic regulatory pathway, making yeast an attractive model for the analysis of this relatively unexplored segment of apoptosis.

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Last edited: February 10, 2006 by KaiFr