Identification of the regions of porcine VCP preventing its function in Saccharomyces cerevisiae.
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 homologue VCP alternates between the endoplasmic reticulum and the centrosome in dependence of the cell cycle. 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
F
F
Physiologisch-chemisches Institut, Universität Tübingen, Germany.
Schlauer
J
J
Fröhlich
K U
KU
D000251:Adenosine Triphosphatases; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D018797:Cell Cycle Proteins / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D005816:Genetic Complementation Test; D008861:Microsomes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013552:Swine; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein