Nuclear import in permeabilized protoplasts from higher plants has unique features.
The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.
Hicks
G R
GR
Michigan State University-Department of Energy, East Lansing 48824-1312, USA.
Smith
H M
HM
Lobreaux
S
S
Raikhel
N V
NV
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D002467:Cell Nucleus / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D028884:Karyopherins; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010539:Permeability; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D010947:Plants, Toxic; D011523:Protoplasts / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana
D006150:Guanine Nucleotides; D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010940:Plant Proteins