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 a nd yeast. To test this model in plants and to identify components of t he import apparatus, we developed a direct in vitro nuclear import ass ay by using tobacco protoplasts that were permeabilized without deterg ents such as digitonin or Triton X-100. Substrates were imported speci fically by a mechanism that required only guanine nucleotides. Moreove r, in vitro import did not require exogenous cytosol. To investigate t his novel finding, we isolated a full-length cDNA encoding an Arabidop sis homolog of vertebrate and yeast nuclear localization signal recept ors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protopla sts, even in the presence of 0.1% Triton X-100, indicating that this f actor and probably others were retained to an extent sufficient to sup port import. The lectin wheat germ agglutinin bound to the nucleus; ho wever, it did not block translocation in our system, indicating that d irect interaction with polysaccharide modifications at the nuclear por e complex was probably not essential for import in plants. Other featu res of in vitro import included reduced but significant import at low temperature.