Ribosomal proteins of Thermus thermophilus fused to beta-galactosidase are imported into the nucleus of eukaryotic cells.
Archaea, Bacteria, and Eukarya have 34 homologous ribosomal protein (RP) families in common. Comparisons of published amino acid sequences prompted us to question whether RPs of the prokaryote Thermus thermophilus contain nuclear localization signals (NLSs), which are recognized by the nuclear import machinery of eukaryotic cells and are thereby translocated into the nucleoplasm ultimately accumulating in the nucleolus. Several RPs of T. thermophilus - specifically S12, S17, and L2 - were selected for this study since their three-dimensional structures as well as rRNA interaction patterns are precisely known at the molecular level. Fusion proteins of these RPs were constructed and subsequently expressed in COS cells. N-terminally tagged fusions with dimeric EGFP and C-terminally tagged hybrids with beta-galactosidase of prokaryotic RP S17 (S17p) were targeted to the nucleoplasm where they were visualized by direct fluorescence and by indirect immune staining, respectively. A region containing the classical monopartite NLS KRKR, which is known to physically interact with karyopherin alpha2, was delineated by tagging specific S17p fragments with beta-galactosidase. Unexpectedly, S12p and L2p hybrids accumulated in the nucleolus. Due to their size, RPs tagged with beta-galactosidase can only be imported into the nucleus when NLS-recognition is mediated by karyopherins since they are otherwise excluded from entry into the nucleoplasm of eukaryotic cells. Our results indicate that after the formation of the nuclear compartment during evolution, the newly established eukaryotic cell relied on the pre-existing basic amino acid clusters of the prokaryotic RPs for use as NLSs.
Perić
Mark
M
Center of Experimental Medicine, Institute of Molecular Cell Biology, Hamburg University, Martinistrasse 52, D-20246 Hamburg, Germany.
Schedewig
Pia
P
Bauche
Andreas
A
Kruppa
Antonina
A
Kruppa
Joachim
J
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002466:Cell Nucleolus / Q000378:metabolism*; D002522:Chlorocebus aethiops; D019143:Evolution, Molecular; D012335:RNA, Ribosomal / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D016963:Thermus thermophilus / Q000235:genetics; D028901:alpha Karyopherins / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D012335:RNA, Ribosomal; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D028901:alpha Karyopherins; C087978:karyopherin alpha 2; D001616:beta-Galactosidase