Mapping of a Sequence Essential for the Nuclear Transport of theXenopusRibosomal Transcription Factor xUBF Using a Simple Coupled Translation-Transport and Acid Extraction Approach

The amino acid sequences directing the nuclear transport of the RNA polymerase I transcription factor xUBF have been studied by a novel combination of in oocyte-coupled translation-nuclear transport and selective HCl extraction. Synthetic mRNA was used to direct the translation of labeled xUBF and its mutants in microinjected oocytes. After manual dissection of nuclei and cytoplasm, labeled xUBF and mutants were isolated essentially pure by HCl extraction. Using deletion mutations, a sequence essential, but not necessarily sufficient, for nuclear transport was mapped to a 29-amino-acid segment lying between the most carboxyterminal putative HMG-box DNA-binding domain, HMG-box 5, and the highly acidic carboxy-terminal domain. It was shown that deletion of only 5 amino acids from this segment eliminated xUBF transport, and it could be deduced that at least 11 of the 29 amino acids were essential for nuclear transport. The segment of xUBF necessary for nuclear transport contains a sequence conforming to the bipartite nuclear transport motif consensus, but this sequence in itself was insufficient for nuclear transport.