LAP2 binds to BAF.DNA complexes: requirement for the LEM domain and modulation by variable regions.
LAP2 belongs to a family of nuclear membrane proteins sharing a 43 residue LEM domain. All LAP2 isoforms have the same N-terminal 'constant' region (LAP2-c), which includes the LEM domain, plus a C-terminal 'variable' region. LAP2-c polypeptide inhibits nuclear assembly in Xenopus extracts, and binds in vitro to barrier-to-autointegration factor (BAF), a DNA-bridging protein. We tested 17 Xenopus LAP2-c mutants for nuclear assembly inhibition, and binding to BAF and BAF small middle dotDNA complexes. LEM domain mutations disrupted all activities tested. Some mutations outside the LEM domain had no effect on binding to BAF, but disrupted activity in Xenopus extracts, suggesting that LAP2-c has an additional unknown function required to inhibit nuclear assembly. Mutagenesis results suggest that BAF changes conformation when complexed with DNA. The binding affinity of LAP2 was higher for BAF small middle dotDNA complexes than for BAF, suggesting that these interactions are physiologically relevant. Nucleoplasmic domains of Xenopus LAP2 isoforms varied 9-fold in their affinities for BAF, but all isoforms supershifted BAF small middle dotDNA complexes. We propose that the LEM domain is a core BAF-binding domain that can be modulated by the variable regions of LAP2 isoforms.
Shumaker
D K
DK
Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 N.Wolfe Street, Baltimore MD 21205, USA.
Lee
K K
KK
Tanhehco
Y C
YC
Craigie
R
R
Wilson
K L
KL
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012996:Solutions; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D008565:Membrane Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D012996:Solutions; C093413:lamina-associated polypeptide 2; D004247:DNA