Identification of the nuclear localization signal of the POU domain protein Tst-1/Oct6.
POU domain proteins are important regulators of development and terminal differentiation based upon their transcriptional activity in the nucleus. Here, we analyzed the mechanism underlying the nuclear localization of Tst-1/Oct6, a member of this family that regulates events during neurogenesis and myelination. Nuclear localization of Tst-1/Oct6 was dependent on the POU domain, as its deletion prevented access to the nucleus, whereas its transfer to the amino terminus of beta-galactosidase was sufficient to prompt nuclear accumulation of this normally cytosolic protein. Interestingly, nuclear localization and high affinity DNA binding were two independent functions of the POU domain and could be separated in several mutants. While specific high affinity binding to DNA required the presence of both the POU-specific and the POU homeodomain, the POU-specific domain was dispensable for nuclear localization of Tst-1/Oct6. Rather, the nuclear localization function was selectively contained within the POU homeodomain. Specifically, a basic cluster (GRKRKKRT) preceding helix 1 of the homeodomain was shown by deletion mutagenesis to be involved in the nuclear localization of Tst-1/Oct6. This sequence, which is highly conserved among POU domain proteins, was by itself capable of translocating beta-galactosidase to the nucleus defining it as the bona fide nuclear localization signal of Tst-1/Oct6 and presumably other POU domain factors.
Sock
E
E
Zentrum für Molekulare Neurobiologie, Universität Hamburg, Martinistrasse 52, D-20246 Hamburg, Federal Republic of Germany.
Enderich
J
J
Rosenfeld
M G
MG
Wegner
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D050816:Octamer Transcription Factor-6; D009838:Oligodeoxyribonucleotides; D011388:Prolactin / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D001616:beta-Galactosidase / Q000096:biosynthesis
D004268:DNA-Binding Proteins; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; C494179:Pou3f1 protein, rat; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D050816:Octamer Transcription Factor-6; D011388:Prolactin; D001616:beta-Galactosidase