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Heterokaryon Technique for Analysis of Cell Type-specific Localization
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A short amino acid sequence able to specify nuclear location.

D Kalderon, B L Roberts, W D Richardson

    Cell
    |December 1, 1984
    PubMed
    Summary

    A specific amino acid sequence, Pro-Lys-Lys-Lys-Arg-Lys-Val, functions as an autonomous nuclear localization signal. This sequence directs proteins, including beta-galactosidase and pyruvate kinase, to accumulate within the cell nucleus.

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    Area of Science:

    • Molecular Biology
    • Cell Biology
    • Protein Localization

    Background:

    • SV40 large T antigen normally accumulates in the nucleus.
    • A specific region around Lys-128 is crucial for this nuclear localization.
    • Mutants lacking this region are found in the cytoplasm.

    Purpose of the Study:

    • To determine if the identified amino acid sequence acts as an autonomous nuclear localization signal.
    • To test the signal's ability to direct cytoplasmic proteins to the nucleus.
    • To characterize the minimal sequence required for nuclear import.

    Main Methods:

    • Constructing fusion proteins of beta-galactosidase and pyruvate kinase with the putative SV40 large T antigen nuclear localization signal.
    • Analyzing the subcellular localization of wild-type and mutant SV40 large T antigen.
    • Mutating key amino acids within the identified signal sequence.

    Main Results:

    • Fusion proteins containing the sequence element accumulated exclusively in the nucleus.
    • The signal's activity was dependent on the specific amino acids, as mutation of Lys-128 to Thr abolished nuclear accumulation.
    • A minimal sequence, Pro-Lys-Lys-Lys-Arg-Lys-Val, was identified as sufficient for nuclear targeting.

    Conclusions:

    • The short amino acid sequence Pro-Lys-Lys-Lys-Arg-Lys-Val functions as a potent nuclear localization signal.
    • This signal can independently direct proteins of diverse origins to the nucleus.
    • The findings suggest this sequence may be a prototype for similar signals in other nuclear proteins.