NuMA: a bipartite nuclear location signal and other functional properties of the tail domain.
Nuclear Mitotic Apparatus protein (NuMA) is a 238-kDa protein of the nuclear matrix in interphase that relocates to the spindle poles in mitosis. The globular tail domain (residues 1701 to 2115) contains the nuclear targeting sequence, the site for binding to the mitotic spindle as well as a site responsible for nuclear reformation. To more precisely map these sites, we inserted full-length human NuMA and 16 derivatives with increasing truncations of the tail domain into the pCMV5 vector and induced transient expression. NuMA was found in the interphase nucleus of all transfected BHK cells expressing either full-length NuMA or NuMA mutant proteins ending at or after residue 2005. In contrast, mutants ending at or before residue 2003 remained in the cytoplasm. In the full-length NuMA molecule, point mutations at position 1988 or 1989 or a double mutation at residues 2004 and 2005 cause NuMA to accumulate in the cytoplasm of both BHK and HeLa cells. The combined results indicate a bipartite nuclear location signal involving the sequences RKR (1987-1989) and KK (2004-2005) which are separated by 14 amino acid residues. In 30% of BHK cells transfected by the full-length clone, cytoplasmic aggregates of NuMA that colocalize with the centrosomes were documented in addition to the nuclear staining. In cells with large aggregates the cytoplasmic microtubular profile was disturbed. Observation of micronuclei formation suggests that a region important for normal nuclear reformation lies in the C-terminal 130 residues. Finally, NuMA mutant proteins ending at or after residue 1800 bound to the spindle poles of mitotic cells, while NuMA proteins ending at or before residue 1750 did not.
Gueth-Hallonet
C
C
Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.
Weber
K
K
Osborn
M
M
D000595:Amino Acid Sequence; D000818:Animals; D034961:Antigens, Nuclear; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D008969:Molecular Sequence Data; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; C050045:NUMA1 protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins