Identification and characterization of nuclear location signal-binding proteins in nuclear envelopes.
A radioiodinated, photoactivable synthetic nonapeptide corresponding to the nuclear location signal (NLS) of SV40 large T antigen has been used in photolabelling reactions with purified mouse liver nuclei, nuclear envelopes and other cellular fractions, to identify specific NLS-binding proteins which may be involved in selective transport of karyophilic proteins. SDS-polyacrylamide gel analysis of photolabelled products demonstrates that a 60 kDa nuclear protein and four nuclear envelope proteins (67, 60, 53 and 47 kDa) bind specifically to the native NLS and not to a mutant NLS or unrelated sequences. This binding shows saturation kinetics, with highest affinity of the NLS for the 60 and 67 kDa proteins. The nuclear 60 kDa NLS-binding protein is identical to the nuclear envelope 60 kDa NLS-binding protein by two-dimensional gel analysis of labelled proteins. Biochemical fractionation of labelled nuclear envelopes suggests that the 53 and 47 kDa proteins are peripheral membrane proteins whereas the 67 and 60 kDa proteins can be localized to the pore complex. The NLS also binds to solubilized 67, 60, 53 and 47 kDa proteins but with decreased affinity. Our results suggest that one of the early steps in selective nuclear transport of proteins may be the recognition of the NLS by the 60 kDa and/or 67 kDa binding proteins present in the nuclear pore complex.
Pandey
S
S
Centre for Cellular and Molecular Biology, Hyderabad, India.
Parnaik
V K
VK
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D002451:Cell Compartmentation; D015180:Electrophoresis, Gel, Two-Dimensional; D066298:In Vitro Techniques; D008099:Liver; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism
D000952:Antigens, Polyomavirus Transforming; D009687:Nuclear Proteins; D010455:Peptides