The nuclear localization signal (NLS) of PDX-1 is part of the homeodomain and represents a novel type of NLS.
The beta-cell homeodomain transcription factor PDX-1 has vital functions both in controlling the expression of pancreatic polypeptide hormones and in the development of the pancreas. The transactivating and DNA-binding properties of PDX-1 have been well characterized, but nuclear transport is still undefined. Here we show that PDX-1 bears a nuclear localization signal (NLS) that is part of helix 3 of the homeodomain. PDX-1 deletion mutants were tagged with enhanced green fluorescent protein (EGFP) and expressed in COS-7 cells. Subcellular localization of the respective PDX-1-EGFP fusion proteins was analyzed by direct fluorescence microscopy and Western immunoblotting using an anti-(GFP). As a result we were able to demonstrate that the homeodomain or helix 3 alone was sufficient and necessary for transport into the nucleus. Point mutations of basic amino acid residues within helix 3 led to identification of an NLS with six amino acids being crucial for nuclear transport of PDX-1. Because this NLS does not match known examples of NLSs, the PDX-1 NLS may represent a novel class of NLS.
Hessabi
B
B
Department of Biochemistry, School of Medicine, Ernst-Moritz-Arndt- University, Greifswald, Germany.
Ziegler
P
P
Schmidt
I
I
Hessabi
C
C
Walther
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D049452:Green Fluorescent Proteins; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics*; D014162:Transfection
D017931:DNA Primers; D018398:Homeodomain Proteins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; D049452:Green Fluorescent Proteins