Sequential recruitment of the mRNA decay machinery to the iron-regulated protein Cth2 in Saccharomyces cerevisiae.

Post-transcriptional factors importantly contribute to the rapid and coordinated expression of the multiple genes required for the adaptation of living organisms to environmental stresses. In the model eukaryote Saccharomyces cerevisiae, a conserved mRNA-binding protein, known as Cth2, modulates the metabolic response to iron deficiency. Cth2 is a tandem zinc-finger (TZF)-containing protein that co-transcriptionally binds to adenine/uracil-rich elements (ARE) present in the 3'-untranslated region of iron-related mRNAs to promote their turnover. The nuclear binding of Cth2 to mRNAs via its TZFs is indispensable for its export to the cytoplasm. Although Cth2 nucleocytoplasmic transport is essential for its regulatory function, little is known about the recruitment of the mRNA degradation machinery. Here, we investigate the sequential assembly of mRNA decay factors during Cth2 shuttling. By using an enzymatic in vivo proximity assay called M-track, we show that Cth2 associates to the RNA helicase Dhh1 and the deadenylase Pop2/Caf1 before binding to its target mRNAs. The recruitment of Dhh1 to Cth2 requires the integrity of the Ccr4-Pop2 deadenylase complex, whereas the interaction between Cth2 and Pop2 needs Ccr4 but not Dhh1. M-track assays also show that Cth2-binding to ARE-containing mRNAs is necessary for the interaction between Cth2 and the exonuclease Xrn1. The importance of these interactions is highlighted by the specific growth defect in iron-deficient conditions displayed by cells lacking Dhh1, Pop2, Ccr4 or Xrn1. These results exemplify the stepwise process of assembly of different mRNA decay factors onto an mRNA-binding protein during the mechanism of post-transcriptional regulation.

Glucose uptake in germinating Aspergillus nidulans conidia: involvement of the creA and sorA genes.

D-Glucose uptake in germinating wild-type Aspergillus nidulans conidia is an energy-requiring process mediated by at least two transport systems of differing affinities for glucose: a low-affinity system (K(m) approximately 1.4 mM) and a high-affinity system (K(m) approximately 16 micro M). The low-affinity system is inducible by glucose; the high-affinity system is subject to glucose repression effected by the carbon catabolite repressor CreA and is absent in sorA3 mutant conidia, which exhibit resistance to L-sorbose toxicity. An intermediate-affinity system (K(m) approximately 400 micro M) is present in sorA3 conidia germinating in derepressing conditions. creA derepressed mutants show enhanced sensitivity to L-sorbose. The high-affinity uptake system appears to be responsible for the uptake of this toxic sugar.