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Evidence that the middle T antigen of polyomavirus interacts with the membrane skeleton
D W Andrews1, J Gupta, G Abisdris
1Department of Biochemistry, McMaster University, Hamilton, Ontario, Canada.
Abstract:
The transforming protein of polyomavirus, middle T antigen, is associated with cellular membranes. We have examined the subcellular location of the middle T antigen in two different cell types by fractionation and detergent phase partitioning. Middle T antigen expressed in human cells by a recombinant adenovirus was detected primarily in the membrane skeleton. Sucrose gradient fractionation revealed that the middle T antigen was associated with complexes with molecular weights of 500,000 to 1,000,000. Several markers for cytoskeleton cofractionate with these complexes, including actin, tubulin, and vimentin. Electron micrographs of membrane skeleton prepared from cells expressing middle T antigen demonstrated that this material contained primarily fibrous structures and was clearly devoid of bilayer membranes. These structures were distinct from the filamentous structures observed in fractions enriched for cytoskeleton. Consistent with a role for membrane skeleton localization in transformation, middle T antigen was detected exclusively in fractions enriched for membrane skeleton in middle T antigen-transformed Rat-2 cells. Our results may resolve the apparent difference between middle T antigen localization as determined by immunomicroscopy and that determined by subcellular fractionation.
Insights
Polyomavirus middle T antigen localizes to the membrane skeleton, not just membranes. This finding clarifies its role in cell transformation and resolves conflicting localization data.
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Polyomavirus middle T antigen is a transforming protein linked to cellular membranes.
- Previous studies showed conflicting data on its precise subcellular localization.
Purpose of the Study:
- To investigate the subcellular localization of polyomavirus middle T antigen.
- To clarify its association with cellular structures and role in transformation.
Main Methods:
- Subcellular fractionation and detergent phase partitioning were used.
- Analysis of human cells expressing middle T antigen via recombinant adenovirus.
- Sucrose gradient fractionation and electron microscopy were employed.
- Examination of middle T antigen-transformed Rat-2 cells.
Main Results:
- Middle T antigen was primarily detected in the membrane skeleton.
- It associated with high molecular weight complexes (500,000–1,000,000 Da) containing cytoskeleton markers (actin, tubulin, vimentin).
- Electron microscopy revealed fibrous structures in the membrane skeleton, distinct from bilayer membranes and other cytoskeletal elements.
- Localization was exclusively in membrane skeleton fractions in transformed cells.
Conclusions:
- Polyomavirus middle T antigen localizes to the membrane skeleton, associated with cytoskeletal components.
- This localization provides a clearer understanding of its role in cellular transformation.
- The findings reconcile discrepancies between immunomicroscopy and subcellular fractionation data.