Nuclear targeting of the maize R protein requires two nuclear localization sequences.
Previous genetic and structural evidence indicates that the maize R gene encodes a nuclear transcriptional activating factor. In-frame carboxyl- and amino-terminal fusions of the R gene to the reporter gene encoding beta-glucuronidase (GUS) were sufficient to direct GUS to the nucleus of the transiently transformed onion (Allium cepa) epidermal cells. Further analysis of chimeric constructs containing regions of the R gene fused to the GUS cDNA revealed three specific nuclear localization sequences (NLSs) that were capable of redirecting the GUS protein to the nucleus. Amino-terminal NLS-A (amino acids 100-109, GDRRAAPARP) contained several arginine residues; a similar localization signal is found in only a few viral proteins. The medial NLS-M (amino acids 419-428, MSERKRREKL) is a simian virus 40 large T antigen-type NLS, and the carboxyl-terminal NLS-C (amino acids 598-610, MISESLRKAIGKR) is a mating type alpha 2 type. NLSs M and C are independently sufficient to direct the GUS protein to the nucleus when it is fused at the amino terminus of GUS, whereas NLS-A fused to GUS partitioned between the nucleus and cytoplasm. Similar partitioning was observed when localization signals NLS-A and NLS-C were independently fused to the carboxy-terminal portion of GUS. A sequential deletion of the localization signals indicated that the amino-terminal and carboxyl-terminal fusions of R and GUS were redirected to the nucleus only when both NLS-A and -M, or NLS-C and -M, were present. These results indicate that multiple localization signals are necessary for nuclear targeting of this protein. The conservation of the localization signals within the alleles of R and similar proteins from other organisms is also discussed.
Shieh
M W
MW
Michigan State University, Department of Energy Plant Research Laboratory, East Lansing 48823-1312.
Wessler
S R
SR
Raikhel
N V
NV
D000490:Allium / Q000235:genetics; D000595:Amino Acid Sequence; D001692:Biological Transport / Q000235:genetics; D002451:Cell Compartmentation / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism*; D004252:DNA Mutational Analysis; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics; D006651:Histocytochemistry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics / Q000378:metabolism*
D009687:Nuclear Proteins; D010940:Plant Proteins; C085200:R protein, Zea mays; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase