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Evidence for transmembrane orientation of acylated simian virus 40 large T antigen
Abstract:
In mKSA cells (a simian virus 40-transformed BALB/c mouse tumor cell line), plasma membrane-associated large T antigen (large T) is found in two subfractions of the plasma membrane; a minor amount of large T is recovered from the Nonidet P-40 (NP-40)-soluble plasma membrane fraction, whereas the majority is tightly bound to a substructure of the plasma membrane, the plasma membrane lamina (PML). Only PML-associated large T is fatty acid acylated (U. Klockmann and W. Deppert, EMBO J. 2:1151-1157, 1983). We have analyzed whether these two forms of plasma membrane-associated large T might differ in features like cell surface expression or metabolic stability. In addition, we have asked whether one of the two large Ts might represent the hypothetic, large T-related protein T* (D. F. Mark and P. Berg, Cold Spring Harbor Symp. Quant. Biol. 44:55-62, 1979). We show that in mKSA cells grown in suspension culture, large T associated with the PML is also exposed on the cell surface. This form of large T, therefore, exhibits properties of a transmembrane protein. Large T in the NP-40-soluble plasma membrane fraction could not be labeled with radioiodine on the cell surface and, for this reason, does not seem to be oriented towards the cell surface. In contrast, when mKSA cells were grown on substratum (culture dish), we found that in these cells both NP-40-soluble large T as well as large T anchored in the PML could be cell surface iodinated. We also have analyzed the plasma membrane association of surface T antigen in mKSA cells grown in a mouse as ascites tumor. In tumor cells, only PML-bound large T is cell surface associated. We conclude that differences in extractibility of cell surface-associated large T most likely depend on cell shape and are not an artifact of cell culture. Both NP-40-soluble and PML-bound large Ts are associated with the plasma membrane in a metabolically stable fashion. Neither of the two large Ts represents T*.
Insights
Large T antigen in mKSA cells is found in two plasma membrane fractions. Cell shape influences whether large T antigen is exposed on the cell surface, but it remains metabolically stable.
Area of Science:
- Molecular and Cellular Biology
- Virology
- Biochemistry
Background:
- Simian virus 40-transformed mKSA cells express plasma membrane-associated large T antigen (large T).
- Large T antigen localizes to two plasma membrane subfractions: Nonidet P-40 (NP-40)-soluble and plasma membrane lamina (PML)-bound.
- Only PML-bound large T is fatty acid acylated, suggesting distinct functional roles.
Purpose of the Study:
- To investigate differences in cell surface expression and metabolic stability between the two large T antigen fractions.
- To determine if either large T fraction corresponds to the hypothetical large T-related protein T*.
Main Methods:
- Analysis of large T antigen localization in mKSA cells cultured in suspension and on substratum.
- Cell surface iodination to assess surface exposure of large T antigen.
- Investigation of large T antigen in ascites tumor cells grown in vivo.
- Metabolic labeling and extraction protocols to evaluate metabolic stability.
Main Results:
- In suspension culture, PML-bound large T is cell surface-exposed, while NP-40-soluble large T is not.
- When grown on substratum or in vivo as ascites tumors, both NP-40-soluble and PML-bound large T can be cell surface-associated, indicating cell shape influences localization.
- Both large T fractions exhibit metabolic stability, and neither corresponds to the T* protein.
Conclusions:
- Cell shape, not cell culture artifacts, dictates the differential cell surface exposure of large T antigen fractions.
- PML-bound large T functions as a transmembrane protein when exposed on the cell surface.
- Both NP-40-soluble and PML-bound large T are metabolically stable components of the plasma membrane in mKSA cells.