Subcellular localization of the 30K protein in TMV-inoculated tobacco protoplasts.

We investigated the intracellular localization of the 30K protein in TMV-inoculated tobacco protoplasts by means of pulse-labeling and pulse-chase experiments with [35S]methionine. Protoplasts were lysed with a nonionic detergent and the extracts were centrifuged to yield soluble and crude nuclear fractions. Most of the 30K protein was found in the crude nuclear fraction. The nuclear fraction was further purified by centrifugation in a step-wise Percoll gradient. Nuclei and the 30K protein were found in the same fractions. The results of pulse-chase experiments indicated that the 30K protein is synthesized in the soluble fraction and then translocated to the crude nuclear fraction. The 30K protein of Ls1, a temperature-sensitive (ts) mutant affecting the cell-to-cell viral transport function, was also found in the nuclei, even at a nonpermissive temperature. These results suggested that the 30K protein has to be localized in the nuclei to function, and that impaired translocation of the 30K protein to the nuclei is not responsible for the ts lesion of mutant Ls1.