Use of a general purpose mammalian expression vector for studying intracellular protein targeting: identification of critical residues in the nuclear lamin A/C nuclear localization signal.
We have constructed a general purpose mammalian expression vector for the study of intracellular protein targeting. The vector, p3PK, facilitates construction of N- and/or C-terminal fusions of an amino acid sequence of interest to the normally cytosolic protein chicken muscle pyruvate kinase (CMPK). The vector has been engineered such that any fusion construct can be subcloned into the versatile pJx omega family of mammalian expression vectors and into pGEX bacterial expression vectors, for the generation of affinity reagents. In this paper, we demonstrate the general utility of p3PK by redirecting CMPK to mitochondria (using the twelve amino acid pre-sequence of yeast cytochrome c oxidase subunit IV) and to the nucleus (using a putative eight amino acid nuclear localization signal from human nuclear lamins A and C). We also report that, contrary to the predictions of previously published work, substitution of a critical residue in the nuclear lamin A/C nuclear localization signal (the equivalent of lysine 128 in the SV40 large T nuclear localization signal) retains nuclear localization, and discuss how amino acid context might affect targeting to the nucleus.
Frangioni
J V
JV
Molecular Medicine Unit, Beth Israel Hospital, Boston, MA 02215.
Neel
B G
BG
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D002645:Chickens / Q000235:genetics; D005813:Genes, Synthetic; D005822:Genetic Vectors; D034904:Lamin Type A; D034882:Lamins; D008322:Mammals / Q000378:metabolism*; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D034904:Lamin Type A; D034882:Lamins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011770:Pyruvate Kinase