The DEAD-box helicase Ded1 from yeast is an mRNP cap-associated protein that shuttles between the cytoplasm and nucleus.
Bottom Line: In addition, we show that Ded1 is genetically linked to these factors.Ded1 comigrates with these proteins on sucrose gradients, but treatment with rapamycin does not appreciably alter the distribution of Ded1; thus, most of the Ded1 is in stable mRNP complexes.We conclude that Ded1 is an mRNP cofactor of the cap complex that may function to remodel the different mRNPs and thereby regulate the expression of the mRNAs.
Affiliation: Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.Show MeSH
Mentions: We next asked where the Ded1-GFP fusion protein was located within the cell. Fluorescence microscopy of living cells revealed that the protein was primarily located within the cytoplasm, but the nuclei showed a faint signal relative to the vacuoles/lysosomes. To verify this, we fixed the cells and then stained them with DAPI to identify the nuclei (Figure 1A and B). These experiments confirmed that Ded1 was largely excluded from the nuclei, but that the faint signals corresponded to the nuclei rather than to the vacuoles. Both plasmid-expressed and integrated Ded1-GFP showed the same distributions, which indicated that the expression levels did not alter the results. However, the intensity of the Ded1-GFP signal varied between cells in both the plasmid-borne and integrated cells. This implied that the expression levels of Ded1 were variable between cells (Figure 1A and B and Supplemental Figure S3). Thus, our results were consistent with the possibility that a small amount of Ded1 was located in the nucleus.
Affiliation: Expression Génétique Microbienne, CNRS FRE3630 (UPR9073), in association with Université Paris Diderot, Sorbonne Paris Cité, Paris 75005, France Université Paris-Sud, Ecole Doctorale 426 GGC, Orsay, France.