The roles of multiple importins for nuclear import of murine aristaless-related homeobox protein.
Nuclear import of proteins with nuclear localization signals (NLSs) is mediated by shuttling carriers, the importins. Some cargoes display more than a single NLS, and among these are homeodomain proteins such as Arx, which is critical for development of multiple tissues. Arx has two functional NLSs. The present studies show that several pathways can import Arx via its NLS2, which is within its DNA binding homeodomain. Using an in vitro nuclear import assay, we show that import of Arx via NLS2 can be mediated by importin beta1, importin 9, or importin 13, with binding being strongest to importin beta1. All binding is sensitive to RanGTP. Experiments based on precise domain deletions indicate that NLS2 binds impbeta1, imp9, and imp13 and includes both an importin binding subdomain and a regulatory subdomain with arginine residues being important for function. Moreover, Arx can be co-precipitated with these importins when NLS2 is present. Although nuclear import of Arx can be mediated by these three importin betas, importin beta1 seems to play the major role judging from in vivo small interfering RNA ablations and the in vitro import assay. This is the first evidence to show the role of importin beta1 in nuclear import of paired-type homeodomain proteins. We propose a novel and possibly quite general mechanism for nuclear import of paired-type homeodomain proteins which is critical for development.
Lin
Wenbo
W
School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China.
Ye
Wenduo
W
Cai
Lanlan
L
Meng
Xinyi
X
Ke
Guifen
G
Huang
Caoxin
C
Peng
Zi
Z
Yu
Yinhua
Y
Golden
Jeffrey A
JA
Tartakoff
Alan M
AM
Tao
Tao
T
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D041681:NIH 3T3 Cells; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D014157:Transcription Factors / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism
C469265:ARX protein, mouse; D018398:Homeodomain Proteins; D028884:Karyopherins; D014157:Transcription Factors; D028961:beta Karyopherins