Identification of a segment of the small nucleolar ribonucleoprotein-associated protein GAR1 that is sufficient for nucleolar accumulation.
GAR1 is a 25-kDa nucleolar protein that is essential for yeast cell growth. The protein is associated with a subset of small nucleolar RNAs and is required for pre-rRNA processing. By expressing in yeast various deletions of GAR1 fused to a reporter protein, we have searched for which particular domain of GAR1 can account for its nucleolar localization. We report here that the glycine/arginine-rich domains of GAR1, which are shared by several other nucleolar proteins, are neither sufficient nor required for the steady-state accumulation of the fusion protein in the nucleolus. We further demonstrate that the central domain of GAR1 is both sufficient to target the beta-galactosidase to the yeast nucleolus and to restore the growth of a strain deficient in GAR1. As opposed to the other characterized nucleolar proteins, the nucleolar targeting domain of GAR1 does not exhibit any homology with the SV40 T-antigen-type nuclear localization sequence. Moreover, none of the modified GAR1 proteins that we examined has allowed us to distinguish the nuclear and nucleolar targeting domains. The presence in GAR1 of a single domain that is responsible for both nuclear entry and nucleolar accumulation suggests that GAR1 either could be carried piggyback by another nucleolar component, possibly as part of a small nucleolar ribonucleoprotein particle, or could be transported to the nucleolus by using a pathway different from the other nucleolar proteins.
Laboratoire de Biologie Moléculaire Eucaryote, Toulouse, France.
J P
JP
Girard
C
C
Bagni
M
M
Caizergues-Ferrer
F
F
Amalric
B
B
Lapeyre
D000595:Amino Acid Sequence; D001483:Base Sequence; D002466:Cell Nucleolus / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D005656:Fungal Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C073180:GAR1 protein, S cerevisiae; D001616:beta-Galactosidase