A Drosophila homolog of the membrane fusion protein CDC48/p97 has been cloned. The open reading frame of the Drosophila homolog encodes a protein 801 amino acids long (TER94), which shows high similarity to the known CDC48/p97 sequences. The chromosomal position of TER94 is 46 C/D. TER94 is expressed in embryo, in pupae and in imago, but is suppressed in larva. In the imago, the immunoreactivity is exclusively present in the head and in the gonads of both sexes. In the head the most striking staining is observed in the entire neuropil of the mushroom body and in the antennal glomeruli. In addition to TER94, sex-specific forms are also detected in the gonads of the imago: p47 in the ovaries and p98 in the testis. TER94/p47 staining is observed in the nurse cells and often in the oocytes, while TER94/p98 staining is present in the sperm bundles. On the basis of its distribution it is suggested that TER94 functions in the protein transport utilizing endoplasmic reticulum and Golgi derived vesicles (Pinter, 1998).

An Arabidopsis thaliana CDC48 gene has been identified which, unlike the putative mammalian homolog vasolin-containing protein (VCP), functionally complements Saccharomyces cerevisiae cdc48 mutants. CDC48 is an essential gene in S. cerevisiae and genetic studies suggest a role in spindle pole body separation. Biochemical studies link VCP function to membrane trafficking and signal transduction. The AtCDC48 expression pattern is described in a multicellular eukaryote; the zones of cell division, expansion and differentiation are physically separated in higher plants, thus allowing the analysis of in situ expression patterns with respect to the state of cell proliferation. AtCDC48 is highly expressed in the proliferating cells of the vegetative shoot, root, floral inflorescence and flowers, and in rapidly growing cells. AtCDC48 mRNA and the encoded protein are up-regulated in the developing microspores and ovules. AtCDC48 expression is down-regulated in most differentiated cell types. AtCDC48p is primarily localized to the nucleus and, during cytokinesis, to the phragmoplast, a site where membrane vesicles are targeted in the deposition of new cell wall materials. This study shows that the essential cell division function of CDC48 has been conserved by, at least, some multicellular eukaryotes and suggests that in higher plants, CDC48 functions in cell division and growth processes (Feiler, 1995).

Yeast mutants of cell cycle gene cdc48-1 arrest as large budded cells with microtubules spreading aberrantly throughout the cytoplasm from a single spindle plaque. The gene was cloned and disruption proved it to be essential. The CDC48 sequence encodes a protein of 92 kD that has an internal duplication of 200 amino acids and includes a nucleotide binding consensus sequence. Vertebrate VCP has a 70% identity over the entire length of the protein. Yeast Sec18p and mammalian N-ethylmaleimide-sensitive fusion protein, which are involved in intracellular transport, yeast Pas1p, which is essential for peroxisome assembly, and mammalian TBP-1, which influences HIV gene expression, are 40% all identical to each outer in the duplicated region. Antibodies against CDC48 recognize a yeast protein of apparently 115 kD and a mammalian protein of 100 kD. Both proteins are bound loosely to components of the microsomal fraction as described for Sec18p and N-ethylmaleimide-sensitive fusion protein. This similarity suggests that CDC48p participates in a cell cycle function related to that of N-ethylmaleimide-sensitive fusion protein/Sec18p in Golgi transport (Frohlich, 1991).

The fusion of endoplasmic reticulum (ER) membranes in yeast is an essential process required for normal progression of the nuclear cell cycle (karyogamy) and the maintenance of an intact organellar compartment. This process requires a novel fusion machinery distinct from the classic membrane docking/fusion machinery containing Sec17p (alpha-SNAP) and Sec18p (NSF). Cdc48p, a cell-cycle protein with homology to Sec18p, is required in ER fusion. A temperature-sensitive cdc48 mutant is conditionally defective in ER fusion in vitro. Addition of purified Cdc48p restores the fusion of isolated cdc48 mutant ER membranes. It is proposed that Cdc48p is part of an evolutionarily conserved fusion/docking machinery involved in multiple homotypic fusion events (Latterich, 1995).

Golgi cisternae regrow in a cell-free system from mitotic Golgi fragments incubated with buffer alone. Pretreatment with NEM or salt washing inhibits regrowth, but this can be restored either by p97, an NSF-like ATPase, or by NSF together with SNAPs and p115, a vesicle docking protein. The morphology of cisternae regrown with p97 and NSF-SNAPs-p115 differs, suggesting that they play distinct roles in rebuilding Golgi cisternae after mitosis (Rabouille, 1995).

Cdc48p is essential for homotypic endoplasmic reticular fusion in Saccharomyces cerevisiae. It is localized at the endoplasmic reticulum during most of the cell division cycle but concentrates in the nucleus at the G1/S-transition. Its mammalian homolog VCP (valosin-containing protein) alternates between the endoplasmic reticulum and the centrosome in in a cell cycle dependent manner. 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 N-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 C-terminal Cdc48p domain for the C-terminus of VCP leads to normal viability of the cell, even at low expression levels (Madeo, 1997).

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homolog VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both 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. 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 (Madeo, 1998).

A library of randomly generated 10 residue peptides fused to the N-terminus of a reporter protein was screened in the yeast Saccharomyces cerevisiae for sequences that can target the reporter for degradation by the N-end rule pathway, a ubiquitin (Ub)-dependent proteolytic system that recognizes potential substrates through binding to their destabilizing N-terminal residues. One of the N-terminal sequences identified by this screen was used in a second screen for mutants incapable of degrading the corresponding reporter fusion. A mutant thus identified had an abnormally low content of free Ub. This mutant was found to be allelic to a previously isolated mutant in a Ub-dependent proteolytic system distinct from the N-end rule pathway. The gene involved, termed UFD3, encodes an 80 kDa protein containing tandem repeats of a motif that is present in many eukaryotic proteins and called the WD repeat. Both co-immunoprecipitation and two-hybrid assays demonstrate that Ufd3p is an in vivo ligand of Cdc48p, an essential ATPase required for the cell cycle progression and the fusion of endoplasmic reticulum membranes. Similar to Ufd3p, Cdc48p is also required for the Ub-dependent proteolysis of test substrates. The discovery of the Ufd3p--Cdc48p complex and the finding that this complex is a part of the Ub system opens up a new direction for studies of the function of Ub in the cell cycle and membrane dynamics (Ghislain, 1996).

The inactivation of the prototype NF-kappaB inhibitor, IkappaBalpha, occurs through a series of ordered processes including phosphorylation, ubiquitin conjugation, and proteasome-mediated degradation. Valosin-containing protein (VCP), an AAA (ATPases associated with a variety of cellular activities) family member, co-precipitates with IkappaBalpha immune complexes. The ubiquitinated IkappaBalpha conjugates readily associate with VCP both in vivo and in vitro, and this complex appears dissociated from NF-kappaB. In ultracentrifugation analysis, physically associated VCP and ubiquitinated IkappaBalpha complexes sediment in the 19 S fractions, while the unmodified IkappaBalpha sediments in the 4.5 S fractions deficient in VCP. Phosphorylation and ubiquitination of IkappaBalpha are critical for VCP binding, which in turn is necessary but not sufficient for IkappaBalpha degradation; while the N-terminal domain of IkappaBalpha is required in all three reactions, both N- and C-terminal domains are required in degradation. Further, VCP co-purifies with the 26 S proteasome on two-dimensional gels and co-immunoprecipitates with subunits of the 26 S proteasome. These results suggest that VCP may provide a physical and functional link between IkappaBalpha and the 26 S proteasome and play an important role in the proteasome-mediated degradation of IkappaBalpha (Dai, 1998).