Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to the stress-inducible yeast protein STI1.
A transformation-sensitive human protein (IEF SSP 3521) that is 2-fold up-regulated in SV40-transformed MRC-5 fibroblasts has been purified by two-dimensional gel electrophoresis, microsequenced, and cDNA cloned using oligodeoxyribonucleotides. The 2.1-kilobase cDNA encodes a 543-amino acid protein with a calculated molecular mass of 62.6 kDa and a calculated pI of 6.77. Expression of the cDNA in AMA cells using the vaccinia virus expression system followed by two-dimensional gel electrophoresis showed that the protein comigrated with IEF SSP 3521. The protein contains the tetratricopeptide repeat found in families of fungal proteins required for mitosis and RNA synthesis. In particular, the protein has 42% amino acid sequence identity to STI1, a stress-inducible mediator of the heat shock response in Saccharomyces cerevisiae. Northern blot analysis indicated that the 3521 mRNA is up-regulated in several transformed cells. Immunofluorescence studies using a polyclonal antibody raised against the purified protein revealed that the antigen is present mainly in the nucleus of SV40 transformed MRC-5 fibroblasts, while it localizes to the Golgi apparatus and small vesicles in their normal counterparts. The possible physiological role of IEF SSP 3521 is discussed in the light of the structural relationship with STI1.
Honoré
B
B
Institute of Medical Biochemistry, Aarhus University, Denmark.
Leffers
H
H
Madsen
P
P
Rasmussen
H H
HH
Vandekerckhove
J
J
Celis
J E
JE
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D005814:Genes, Viral; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014170:Transformation, Genetic; D015854:Up-Regulation; D014616:Vaccinia virus / Q000235:genetics
D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C079373:STIP1 protein, human; C073597:TPR protein, human; D004247:DNA