SV40 T ANTIGEN: MODEL FOR NUCLEAR TRANSPORT OF PROTEINS

The mechanism by which proteins are transported to the nucleus
in eucaryotic cells will be examined. Elucidation of the
mechanism(s) governing nuclear transport and nucleocytoplasmic
interactions has broad implications to understanding the regulation
of gene expression, oncogenic transformation, and the control of
early embryonic development. The simiam virus 40 (SV40) large
tumor (T) antigen offers an exceptional model for the analysis of
nuclear transport; the T antigen polypeptide is well characterized,
the role of T antigen in replication and oncogenic transformation
is well established, the nuclear transport signal of T antigen is
known, and the T antigen polypeptide is localized at both the
nucleus and the cell surface. A system for the analysis of nuclear
transport has been developed which utilizes microinjection of
mammalian cells with synthetic peptide nuclear transport signals
crosslinked to carrier proteins. A peptide homologous to the T
antigen nuclear transport signal induces the nuclear transport of
carrier proteins, but no transport occur when proteins are coupled
to a synthetic peptide homologous to a nuclear-transport
defective T antigen. The first specific aim will be to examine
modified T antigen nuclear transport signal peptides, as well as
signal peptides from other proteins for induction of nuclear
transport. The flexibility of the nuclear transport machinery will
be evaluated by measuring the kinetics of transport for modified
peptides, and competitive microinjection experiments will
determine whether different cellular factors are involved in
recognition of dissimilar transport signals from other proteins.
Second, the cellular protein that binds to the T antigen transport
signal will be isolated and characterized. The signal peptide and
anti-idiotypic antibodies that mimic the conformation of the
signal peptide will be used to isolate cellular transport factors.
The third specific aim will be to isolate and sequence the gene
encoding the cellular factor that binds the T antigen signal
sequence. The cloned gene will be used to analyze the
conservation of the gene during eucaryotic evolution and to
construct expression vectors. Fourth, a detailed immunological
characterization of the ligand-receptor (signal sequence-transport
factor) interaction will be performed. Finally, an attempt will be
made to reconstitute an in vitro nuclear transport system using
isolated nuclear transport factors.