Human heat shock factor 1 is predominantly a nuclear protein before and after heat stress.
The induction of the heat shock genes in eukaryotes by heat and other forms of stress is mediated by a transcription factor known as heat shock factor 1 (HSF1). HSF1 is present in unstressed metazoan cells as a monomer with low affinity for DNA, and upon exposure to stress it is converted to an 'active' homotrimer that binds the promoters of heat shock genes with high affinity and induces their transcription. The conversion of HSF1 to its active form is hypothesized to be a multistep process involving physical changes in the HSF1 molecule and the possible translocation of HSF1 from the cytoplasm to the nucleus. While all studies to date have found active HSF1 to be a nuclear protein, there have been conflicting reports on whether the inactive form of HSF is predominantly a cytoplasmic or nuclear protein. In this study, we have made antibodies against human HSF1 and have reexamined its localization in unstressed and heat-shocked human HeLa and A549 cells, and in green monkey Vero cells. Biochemical fractionation of heat-shocked HeLa cells followed by western blot analysis showed that HSF1 was mostly found in the nuclear fraction. In extracts made from unshocked cells, HSF1 was predominantly found in the cytoplasmic fraction using one fractionation procedure, but was distributed approximately equally between the cytoplasmic and nuclear fractions when a different procedure was used. Immunofluorescence microscopy revealed that HSF1 was predominantly a nuclear protein in both heat shocked and unstressed cells. Quantification of HSF1 staining showed that approximately 80% of HSF1 was present in the nucleus both before and after heat stress. These results suggest that HSF1 is predominantly a nuclear protein prior to being exposed to stress, but has low affinity for the nucleus and is easily extracted using most biochemical fractionation procedures. These results also imply that HSF1 translocation is probably not part of the multistep process in HSF1 activation for many cell types.
Department of Zoology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6.
P A
PA
Mercier
N A
NA
Winegarden
J T
JT
Westwood
D000818:Animals; D000906:Antibodies; D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006367:HeLa Cells; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D018869:Heat-Shock Response / Q000502:physiology*; D006801:Humans; D013696:Temperature; D014157:Transcription Factors; D014709:Vero Cells
D000906:Antibodies; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; C000619706:HSF1 protein, human; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D014157:Transcription Factors; D016650:Fluorescein-5-isothiocyanate