A Tudor protein with multiple SNc domains from pea seedlings: cellular localization, partial characterization, sequence analysis, and phylogenetic relationships.
A major high molecular weight protein (HMP) in the cytoskeletal fraction from pea has been purified. A combination of chromatographic techniques and protease fragment analysis also facilitated the isolation of the encoding cDNA, disclosing the sequence of the complete open reading frame. The protein possesses four complete N-terminal Staphylococcal nuclease (SNc) domains, a central Tudor domain and a partial SNc domain at the C-terminus, which may act as a coiled-coil cytoskeleton interaction motif. Cell fractionation studies showed that the protein was abundant in the cytoskeleton fraction in dark-grown pea seedlings, but essentially was absent from the nucleus. Gel filtration column chromatography indicated that the native protein exists as a dimer, while isoelectric focusing suggested that there were at least four HMP isotypes. The protein co-eluted with ribosomes from a heparin affinity column in vitro, consistent with ribosome/polysome interactions in vivo. Significantly, sequence analysis of the C-terminal SNc motif may accurately predict nuclear versus cytoplasmic localization resulting in potentially very different functional roles for this protein family in different organisms. An antibody to HMP from peas was also raised and an HMP with a similar molecular mass was detected in the cytoskeleton fractions and to a lesser extent in the nuclear fraction (250 g pellet) from rice and wheat seedlings.
Abe
Shunnosuke
S
Laboratory of Molecular Cell Biology, Department of Biological Resources, Faculty of Agriculture, Ehime University, Matsuyama, 790-8566, Japan. abe@mcb.agr.ehime-u.ac.jp
Sakai
Masako
M
Yagi
Kosaku
K
Hagino
Takehiko
T
Ochi
Katsumasa
K
Shibata
Koichi
K
Davies
Eric
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D008836:Micrococcal Nuclease / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012275:Oryza / Q000235:genetics / Q000378:metabolism; D018532:Pisum sativum / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein / Q000379:methods; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000235:genetics / Q000378:metabolism
D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D010940:Plant Proteins; D016601:RNA-Binding Proteins; D008836:Micrococcal Nuclease