Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding.
The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.
Milarski
K L
KL
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208.
Morimoto
R I
RI
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002466:Cell Nucleolus / Q000378:metabolism*; D002872:Chromosome Deletion; D005455:Fluorescent Antibody Technique; D005796:Genes; D005822:Genetic Vectors; D006367:HeLa Cells / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009154:Mutation; D010446:Peptide Fragments / Q000302:isolation & purification; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014162:Transfection
D006360:Heat-Shock Proteins; D010446:Peptide Fragments; D000255:Adenosine Triphosphate