Mechanism and a peptide motif for targeting peripheral proteins to the yeast inner nuclear membrane.
Trm1 is a tRNA specific m(2)(2)G methyltransferase shared by nuclei and mitochondria in Saccharomyces cerevisiae. In nuclei, Trm1 is peripherally associated with the inner nuclear membrane (INM). We investigated the mechanism delivering/tethering Trm1 to the INM. Analyses of mutations of the Ran pathway and nuclear pore components showed that Trm1 accesses the nucleoplasm via the classical nuclear import pathway. We identified a Trm1 cis-acting sequence sufficient to target passenger proteins to the INM. Detailed mutagenesis of this region uncovered specific amino acids necessary for authentic Trm1 to locate at the INM. The INM information is contained within a sequence of less than 20 amino acids, defining the first motif for addressing a peripheral protein to this important subnuclear location. The combined studies provide a multi-step process to direct Trm1 to the INM: (i) translation in the cytoplasm; (ii) Ran-dependent import into the nucleoplasm; and (iii) redistribution from the nucleoplasm to the INM via the INM motif. Furthermore, we demonstrate that the Trm1 mitochondrial targeting and nuclear localization signals are in competition with each other, as Trm1 becomes mitochondrial if prevented from entering the nucleus.
Lai
Tsung-Po
TP
Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.
Stauffer
Karen A
KA
Murthi
Athulaprabha
A
Shaheen
Hussam H
HH
Peng
Gang
G
Martin
Nancy C
NC
Hopper
Anita K
AK
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D009685:Nuclear Envelope / Q000201:enzymology / Q000378:metabolism*; D019913:Nuclear Localization Signals; D022022:Nuclear Pore / Q000201:enzymology / Q000378:metabolism; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; C506819:Trm1-11, S cerevisiae; D012359:tRNA Methyltransferases; D001616:beta-Galactosidase; D020931:ran GTP-Binding Protein