Nuclear targeting determinants of the phage P1 cre DNA recombinase.
The Cre DNA recombinase of bacteriophage P1 has become a useful tool for genomic manipulation in mice and other eukaryotes. Because Cre is of prokaryotic origin, the 38 kDa protein has been presumed to gain access to the eukaryotic nucleus simply because it is sufficiently small to pass through the nuclear pore by passive diffusion. Instead, we show here that Cre carries nuclear targeting determinants that efficiently direct Cre entry into the nucleus of mammalian cells. Fusions of Cre with green fluorescent protein (GFP) identified two regions that are necessary for nuclear localization. Region I contains a cluster of basic amino acids that is essential for nuclear localization and which resembles a bipartite-like nuclear localization signal. Region II exhibits a beta-sheet structure with which the bipartite motif may interact. However, neither region is by itself sufficient for nuclear localization. Nuclear transport in vitro with a 98 kDa GFP-Cre fusion protein shows that Cre does not gain access to the nucleus by passive diffusion, but instead enters the nucleus by means of an energy-dependent process. Thus, Cre is one of the few prokaryotic proteins that have been shown to carry determinants that allow it to target the eukaryotic nucleus.
Le
Y
Y
Developmental Biology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Gagneten
S
S
Tombaccini
D
D
Bethke
B
B
Sauer
B
B
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017101:Bacteriophage P1 / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism*; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; C045073:Cre recombinase; D019426:Integrases