Both the CDC48 protein in yeast and its homologue VCP in mammalian cells are relocalized during the course of the cell cycle. In both types of cells, the proteins are located at vesicular structures within the cytoplasm, most probably the ER, during most of the cell cycle. However, in human WISH cells and rat hepatocytes, VCP is concentrated at the centrosomes during the entire mitosis, while in yeast cells Cdc48p accumulates within the nucleus for a short period at START. The temporary concentration of VCP at the centrosomes, in addition to the defect in spindle pole body duplication of arrested cdc48
mutants, suggests a role of VCP/Cdc48p in the proliferation of the spindle-organizing center. The divergent behavior of the highly similar proteins may be due to the different localization of their respective targets. While the centrosomes are freely accessible in the cytosol of mammalian cells, the yeast spindle pole is integrated in the nuclear envelope, which remains intact during the whole cell cycle. We assume that Cdc48p can only reach its target site at the spindle pole body from within the nucleus. The accumulation of the protein within the nucleus may eliminate the need for a specific targeting of Cdc48p to the spindle pole body. A similar behavior has been described for Dsk2p, which is involved in spindle pole duplication and is found inside the nucleus, but not attached to the spindle pole body (Biggins et al., 1996
). The defined moments of relocalization during the cell cycle are in accordance with the role for Cdc48p and VCP proposed by us: the centrosome is duplicated during mitosis, while the spindle pole body is duplicated in late G1.
Our results suggest two cellular functions of both Cdc48p and VCP: an essential role in the cell division cycle, probably at the spindle pole body respectively the centrosome, and a function in the homotypic fusion of the ER. The two functions do not necessarily imply diverse molecular activities. It seems at least as likely that the difference lies primarily in the targets, while the molecular processes may be similar.
The amino-terminal region of Cdc48p contains a functional nuclear targeting consensus sequence as we demonstrated by site-directed mutagenesis. Elimination of the sequence not only prevents nuclear import, but makes a high accumulation of the mutated protein necessary for cell growth. Cell cycle progression apparently requires the presence of Cdc48p in the nucleus. On overexpression of the mutated gene, a small amount of the protein sufficient for its cell cycle function probably enters the nucleus, perhaps due to a marginal nuclear targeting activity of either the few basic residues of the mutated targeting signal or of another stretch of basic amino acid residues (residues 69–74). VCP lacks a nuclear targeting sequence in the corresponding region (see Table ), presumably because the centrosomes have become directly accessible during evolution, making the nuclear import unnecessary.
The relocation of both VCP and Cdc48p at a specific point in the cell cycle requires a regulator. A good candidate is the phosphorylation of a tyrosine residue at the penultimate carboxy-terminal position of VCP, which is triggered by stimulation of the T cell antigen receptor in cultivated murine T cells (Egerton et al., 1992
; Egerton and Samelson, 1994
We demonstrate that Cdc48p is tyrosine phosphorylated depending on the cell cycle phase. Phosphorylation occurs during the same period as nuclear localization of Cdc48p (late G1). A mutation of the tyrosine residue of Cdc48p near the carboxy terminus to phenylalanine, mimicking a nonphosphorylated tyrosine residue, abolishes tyrosine phosphorylation. The mutation has similar effects as the exchange of the nuclear targeting sequence: loss of nuclear import and growth defects when the gene is only expressed at low levels. In contrast, a mutation of the tyrosine residue to glutamic acid, which is the best imitation of a phosphotyrosine possible with the proteinogenic amino acids, results in a concentration of the protein in the nucleus. The abnormal morphology of the nucleus in the cdc48Y834E strain may be an effect of a lack of Cdc48p at the endoplasmic reticulum, resulting either in too little homotypic fusion activity, which would otherwise “clean up the odds and ends,” or in a defective protein coating of the endoplasmic reticulum membrane lacking the (otherwise rather abundant) Cdc48p. This morphological abnormality seems to have little effect on cell proliferation. The generation time is the same as that of wild-type cells.
In contrast to mammals, S. cerevisiae
lacks dedicated tyrosine kinases but contains several “dual specificity” protein kinases which in vitro phosphorylate serine, threonine, and tyrosine residues (Hunter and Plowman, 1997
). In vivo, tyrosine phosphorylation of S. cerevisiae
has been shown in few examples, in addition to the autophosphorylation of some protein kinases (e.g., Spk1p [Stern et al., 1991
] or Mck1p [Lim et al., 1993
]) only for Cdc28p (Booher et al., 1993
), mitogen-activated protein kinases (Errede et al., 1993
), and immunophilin (Wilson et al., 1995
). Thus, Cdc48p is the second example of a physiologically tyrosine phosphorylated S. cerevisiae
protein other than a protein kinase, and, as for Cdc28p and the mitogen-activated protein kinases, tyrosine phosphorylation is phylogenetically conserved between yeast and vertebrates.
The regulation of nuclear import by a protein phosphorylation is a well established phenomenon (see Vandromme et al., 1996
, for review). However, in most cases described, the phosphorylation site (at either serine or threonine residues) is in the vicinity of the nuclear targeting sequence, and the effect of the phosphorylation is a prevention of nuclear import (Moll et al., 1991
; Sidorova et al., 1995
; Tagawa et al., 1995
). The probable explanation of this effect is a neutralization or partial shielding of the positive charges of the nuclear targeting consensus by the negative charges of the phosphoric acid groups. In the case of Cdc48p, the phosphorylated site is a tyrosine residue, it is at the other end of the protein chain, and the effect of the phosphorylation is an induction of nuclear transport. Assuming a principally similar mechanism for the inactivation of the nuclear targeting, the stretch of acidic residues near the phosphorylation site might be able to mask the charge of the nuclear targeting sequence, provided that both elements are adjacent in the folded protein. A deletion of the acidic residues does indeed restore nuclear transport even in Cdc48pY834F
. Nuclear transport of the double mutated protein is incomplete, indicating that the deletion has additional effects besides unmasking the nuclear targeting sequence, e.g., resulting in some misfolded protein accumulating in the cytosol. Two-hybrid experiments do not show a direct affinity of the parts of Cdc48p containing the nuclear targeting signal and the carboxy-terminal region, but both regions interact with the central part of the protein. The interaction of the carboxy terminus is dependent on residue 834: in the wild-type form, it shows little interaction with the central part of the protein; in case of the nonphosphorylatable phenylalanine, the interaction is stronger, while no interaction is found if residue 834 is a glutamic acid, mimicking a phosphotyrosine. We suggest that in the nonphosphorylated form of Cdc48p, attachment of the region of the nuclear targeting signal and of the carboxy terminus to the core of the protein brings them close to one another, resulting in a masking of the targeting sequence by the stretch of acidic residues. Tyrosine phosphorylation may result in a conformational change releasing the carboxy terminus and exposing the targeting sequence (Figure ). While direct data about the three-dimensional structure of Cdc48p is not available, the idea of a conformational change induced by the phosphorylation is supported by the observation that the phenylalanine mutant form shows an additional band in the protein degradation pattern compared with the wild-type and glutamic acid forms of Cdc48p (not shown), probably due to exposition of an otherwise protected target for proteolysis.
Figure 6 Model of the molecular regulation of Cdc48p nuclear targeting by tyrosine phosphorylation. Affinity (green arrows) of the amino-terminal and the carboxy-terminal elements with the central region bring nuclear targeting signal and acidic domain in proximity, (more ...)
Our observation that some tyrosine phosphorylation can be detected in Cdc48pY834E
, but not in Cdc48pY834F
, is another indication of a structural difference between the phosphorylated and nonphosphorylated form of Cdc48p. Egerton and Samelson (1994)
found some tyrosine phosphorylation of VCP at residues other than tyrosine 804. At least for Cdc48p, these additional phosphorylation events apparently only take place if residue 834 carries a negative charge, indicating an exposition of previously hidden residues due to an altered conformation.
It could be argued that the attachment of Cdc48p to cytoplasmic membranes might prevent its nuclear import, and that phosphorylation of tyrosine834 or its exchange to a glutamic acid residue might result in a release from the membranes. However, subcellular fractionation of a Cdc48pY834E strain shows that membrane attachment of the protein is not affected. While 80% of Cdc48pY834E is found in the nuclear fraction, only traces are found in the 170,000 × g supernatant while 20% of Cdc48pY834E is attached to the microsomes.
If tyrosine phosphorylation of Cdc48p is assumed to be the signal for nuclear targeting, the question arises what is the function of the tyrosine phosphorylation in mammalian VCP. Probably, the signal performs the corresponding role, directing the protein to the centrosomes. The colocalization of VCP with the antiphosphotyrosine signal in mitotic WISH cells could be interpreted as an indication, although it does not necessarily mean that both antibodies recognize the same protein. However, while components of both the centrosome (Wickramasinghe and Albertini, 1992
) and the spindle pole body (Donaldson and Kilmartin, 1996
; Friedman et al., 1996
) are phosphorylated in a cell cycle-dependent manner, no tyrosine-phosphorylated protein has yet been described in these organelles.
VCP is the major tyrosine-phosphorylated protein in proliferating cells (Egerton et al., 1992
). A Western blot of synchronized WISH cells indicates that the tyrosine-phosphorylated form of VCP is almost restricted to mitosis, which supports the idea that phosphorylation of VCP and its centrosomal localization might be linked.
Different anti-phosphotyrosine antibodies seem to recognize only a fraction of tyrosine-phosphorylated proteins, probably due to effects of neighboring residues, which makes them more protein specific. This additional specificity or the high abundance of Cdc48p may also be the reason why we have not detected any other tyrosine-phosphorylated protein in our Western blotting of yeast.