Topogenesis of a nucleolar protein: determination of molecular segments directing nucleolar association.
To identify the element(s) in nucleolar proteins which determine nucleolus-specific topogenesis, we have used different kinds of cDNA constructs encoding various chimeric combinations of mutants of the constitutive nucleolar protein NO38 (B23): 1) with an amino terminally placed short "myc tag"; 2) with two different carboxyl terminally attached large alpha-helical coiled coil structures, the lamin A rod domain or the rod domain of vimentin; 3) with the sequence-related nucleoplasmic histone-binding protein nucleo-plasmin; and 4) with the soluble cytoplasmic protein pyruvate kinase. To avoid the problem of formation of complexes with endogenous wild-type (wt) molecules and "piggyback" localization, special care was taken to secure that the mutants and chimeras used did not oligomerize as is typical of protein NO38 (B23). Using microinjection and transfection of cultured cells, we found that the segment comprising the amino-terminal 123 amino acids (aa) alone was sufficient to effect nucleolar accumulation of the construct molecules, including the chimeras with the entire rod domains of lamin A and vimentin. However, when the amino-terminal 109 aa were deleted, the molecules still associated with the nucleolus. The results of further deletion experiments and of domain swaps with nucleoplasmin all point to the topogenic importance of two independent molecular regions located at both the amino- and carboxyl-terminal end. Our definition of dominant elements determining the nucleolar localization of protein NO38 (B23) as well as of diverse nonnucleolar proteins will help to identify its local binding partner(s) and functions, the construction of probes examining other proteins or sequence elements within the nucleolar microenvironment, and the generation of cells with an altered nuclear architecture.
Zirwes
R F
RF
Division of Cell Biology, German Cancer Research Center, Heidelberg, Federal Republic of Germany.
Kouzmenko
A P
AP
Peters
J M
JM
Franke
W W
WW
Schmidt-Zachmann
M S
MS
D000818:Animals; D001665:Binding Sites; D002466:Cell Nucleolus / Q000378:metabolism*; D004252:DNA Mutational Analysis; D000939:Epitopes; D006801:Humans; D034904:Lamin Type A; D034882:Lamins; D008845:Microinjections; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000090243:Nucleophosmin; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D000939:Epitopes; D034904:Lamin Type A; D034882:Lamins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000090243:Nucleophosmin