Carboxyl-terminal basic amino acids in the X domain are essential for the nuclear import of phospholipase C delta1.
BACKGROUND
Although phospholipase C (PLC)delta1 containing a functional nuclear export signal (NES) is normally localized at the plasma membrane and in the cytoplasm, it shuttles between the nucleus and the cytoplasm. Since nucleocytoplasmic shuttling of a molecule is generally regulated by a balance between its NES and the nuclear localization signal (NLS), we examined whether PLCdelta1 contains an NLS sequence.


RESULTS
A region corresponding to the C terminus of the X domain and the XY-linker, which contains clusters of basic amino acid residues, was essential for the nuclear import of PLCdelta1 in Madin-Darby canine kidney cells. A series of point mutations on lysine residues in this region revealed that K432 and K434 in combination were important for the nuclear import. A short synthetic peptide corresponding to residues 429-442, however, was not able to function as an NLS sequence when they were injected into the cytoplasm in a carrier-conjugated form. Neither a longer peptide equivalent to PLCdelta1 412-498 fused to a protein tag consisting of glutathione S-transferase and green fluorescent protein was imported to the nucleus after microinjection into the cytoplasm.


CONCLUSIONS
The nuclear import of PLCdelta1 requires the C-terminus of the X domain, particularly the amino acid residues K432 and K434, and the XY-linker. The region alone, however, cannot serve as a functional NLS. The machinery for nuclear transport may require additional structural component(s) of the enzyme.
Okada
Masashi
M
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo 678-1297, Japan.
Fujii
Makoto
M
Yamaga
Masaki
M
Sugimoto
Hiroaki
H
Sadano
Hiroyuki
H
Osumi
Takashi
T
Kamata
Hideaki
H
Hirata
Hajime
H
Yagisawa
Hitoshi
H
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004285:Dogs; D049452:Green Fluorescent Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008845:Microinjections; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D054803:Phospholipase C delta; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D010738:Type C Phospholipases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007527:Isoenzymes; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D008239:Lysine