No abstract

In Saccharomyces cerevisiae, the rapamycin-sensitive TOR kinases negatively regulate the type 2A-related phosphatase SIT4 by promoting the association of this phosphatase with the inhibitor TAP42. Here, we describe TIP41, a conserved TAP42-interacting protein involved in the regulation of SIT4. Deletion of the TIP41 gene confers rapamycin resistance, suppresses a tap42 mutation, and prevents dissociation of SIT4 from TAP42. Furthermore, a TIP41 deletion prevents SIT4-dependent events such as dephosphorylation of the kinase NPR1 and nuclear translocation of the transcription factor GLN3. Thus, TIP41 negatively regulates the TOR pathway by binding and inhibiting TAP42. The binding of TIP41 to TAP42 is stimulated upon rapamycin treatment via SIT4-dependent dephosphorylation of TIP41, suggesting that TIP41 is part of a feedback loop that rapidly amplifies SIT4 phosphatase activity under TOR-inactivating conditions.

Iron regulatory proteins (IRP1 and IRP2) are RNAbinding proteins that bind to specific structures, termed iron-responsive elements (IREs), that are located in the 5-or 3-untranslated regions of mRNAs that encode proteins involved in iron homeostasis. IRP1 and IRP2 RNA binding activities are regulated by iron; IRP1 and IRP2 bind IREs with high affinity in iron-depleted cells and with low affinity in iron-repleted cells. The decrease in IRP1 RNA binding activity occurs by a switch between apoprotein and 4Fe-4S forms, without changes in IRP1 levels, whereas the decrease in IRP2 RNA binding activity reflects a reduction in IRP2 levels. To determine the mechanism by which iron decreases IRP2 levels, we studied IRP2 regulation by iron in rat hepatoma and human HeLa cells. The iron-dependent decrease in IRP2 levels was not due to a decrease in the amount of IRP2 mRNA or to a decrease in the rate of IRP2 synthesis. Pulse-chase experiments demonstrated that iron resulted in a 3-fold increase in the degradation rate of IRP2. IRP2 degradation depends on protein synthesis, but not transcription, suggesting a requirement for a labile protein. IRP2 degradation is not prevented by lysosomal inhibitors or calpain II inhibitors, but is prevented by inhibitors that block proteasome function. These data suggest the involvement of the proteasome in iron-mediated IRP2 proteolysis. Iron regulatory proteins (IRPs)1 are cytosolic RNA-binding proteins that regulate the post-transcriptional expression of genes that are involved in iron homeostasis (1-4). IRPs were formerly known as the iron-responsive element-binding protein (IRE-BP), the ferritin repressor protein (FRP), and the iron regulatory factor (IRF). IRPs bind with high affinity to RNA stem-loops known as iron-responsive elements (IREs). IREs are located in the 5Ј-untranslated regions of ferritin and erythroid ␦-aminolevulinic acid synthase mRNAs where binding causes translational repression (5-7). Five IREs are located in the 3Ј-untranslated region of transferrin receptor mRNA (8, 9), and binding of the IRP stabilizes transferrin receptor mRNA (9, 10).Two distinct IRPs have been cloned and characterized in mammalian cells and have been designated as IRP1 and IRP2. IRP1 has been cloned from a variety of mammalian species (5,11,12). IRP1 has a molecular mass of 98,000 Da and shares 30% amino acid identity with the 4Fe-4S enzyme, mitochondrial aconitase (13). The 18 active site residues in mitochondrial aconitase, including the 3 cysteines that serve as ligands for the 4Fe-4S cluster are conserved in IRP1 (13). In addition, IRP1 is an active cytosolic aconitase (14, 15). In iron-repleted cells, IRP1 exhibits aconitase activity and contains iron, but binds the IRE with low affinity. In contrast, in iron-depleted cells, IRP1 lacks aconitase activity and iron, but binds the IRE with high affinity. UV cross-linking studies have shown overlap between RNA binding and the aconitase active sites, indicating that RNA binding and aconitase activities are mutually exclusive (16,17). Recent d...

Serum induces Candida albicans to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated theRAS1 gene from C. albicans by complementation. The Candida Ras1 protein, like Ras1 and Ras2 ofSaccharomyces cerevisiae, has a long C-terminal extension. Although RAS1 appears to be the onlyRAS gene present in the C. albicans genome, strains homozygous for a deletion of RAS1(ras1-2/ras1-3) are viable. The Candidaras1-2/ras1-3 mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant activeRAS1V13 allele manifest enhanced hyphal growth, whereas those expressing a dominant negativeRAS1A16 allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in C. albicans through a Ras-mediated signal transduction pathway.