A. P. Mitchell

RIM101-Dependent and -Independent Pathways Govern pH Responses in Candida albicans

ABSTRACT Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101,RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1and PHR1, and repression of PHR2. We find thatRIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation inC. albicans through both RIM101-dependent andRIM101-independent pathways.

A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae.

Two signals activate meiosis in yeast: starvation and expression of the a1 and alpha 2 products of the mating-type locus. Prior studies suggest that these signals stimulate expression of an activator of meiosis, the IME1 (inducer of meiosis) product. We have cloned a gene, IME2, with properties similar to those of IME1: both genes are required for meiosis, and both RNAs are induced in meiotic cells. Elevated dosage of IME1 or IME2 stimulates the meiotic recombination pathway without starvation; thus, the IME products may be part of the switch that activates meiosis. IME1 was found to be required for IME2 expression, and a multicopy IME2 plasmid permitted meiosis in an ime1 deletion mutant. Accordingly, we propose that the IME1 product stimulates meiosis mainly through activation of IME2 expression.

Bipartite structure of an early meiotic upstream activation sequence from Saccharomyces cerevisiae.

Diploid a/alpha Saccharomyces cerevisiae cells cease mitotic growth and enter meiosis in response to starvation. Expression of meiotic genes depends on the IME1 gene product, which accumulates only in meiotic cells. We report here an analysis of the regulatory region of IME2, an IME1-dependent meiotic gene. Deletion and substitution studies identified a 48-bp IME1-dependent upstream activation sequence (UAS). Activity of the UAS also requires the RIM11, RIM15, and RIM16 gene products, which are required for expression of the chromosomal IME2 promoter and for meiosis. Through a selection for suppressors that permit UAS activity in an ime1 deletion mutant, we identified recessive mutations in three genes, SIN3 (also called RPD1, UME4, and SDI1), RPD3, and UME6 (also called CAR80), that were previously known as negative regulators of other early meiotic genes. Mutational analysis of the IME2 UAS reveals two critical sequence elements: a G+C-rich sequence (called URS1), previously identified at many meiotic genes, and a newly described element, the T4C site, that we found at a subset of meiotic genes. In agreement with prior studies, URS1 mutations lead to elevated IME2 UAS activity in the absence of IME1. However, the URS1 mutations prevent any further stimulation of UAS activity by IME1. Repression through URS1 has been shown to require the UME6 gene product. We find that activation of the IME2 UAS by IME1 also requires the UME6 gene product. Thus, UME6 and the URS1 site both have dual negative and positive roles at the IME2 UAS. We propose that IME1 modifies UME6 to convert it from a negulator to a positive Regulor.

Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.

Stimulation of yeast meiotic gene expression by the glucose-repressible protein kinase Rim15p.

The Saccharomyces cerevisiae RIM15 gene was identified previously through a mutation that caused reduced ability to undergo meiosis. We report here an analysis of the cloned RIM15 gene, which specifies a 1,770-residue polypeptide with homology to serine/threonine protein kinases. Rim15p is most closely related to Schizosaccharomyces pombe cek1+. Analysis of epitope-tagged derivatives indicates that Rim15p has autophosphorylation activity. Deletion of RIM15 causes reduced expression of several early meiotic genes (IME2, SPO13, and HOP1) and of IME1, which specifies an activator of early meiotic genes. However, overexpression of IME1 does not permit full expression of early meiotic genes in a rim15delta mutant. Ime1p activates early meiotic genes through its interaction with Ume6p, and analysis of Rim15p-dependent regulatory sites at the IME2 promoter indicates that activation through Ume6p is defective. Two-hybrid interaction assays suggest that Ime1p-Ume6p interaction is diminished in a rim15 mutant. Glucose inhibits Ime1p-Ume6p interaction, and we find that Rim15p accumulation is repressed in glucose-grown cells. Thus, glucose repression of Rim15p may be responsible for glucose inhibition of Ime1p-Ume6p interaction.

Analysis of RIM11, a yeast protein kinase that phosphorylates the meiotic activator IME1.

Many yeast genes that are essential for meiosis are expressed only in meiotic cells. Known regulators of early meiotic genes include IME1, which is required for their expression, and SIN3 and UME6, which prevent their expression in nonmeiotic cells. We report here the molecular characterization of the RIM11 gene, which we find is required for expression of several early meiotic genes. A close functional relationship between RIM11 and IME1 is supported by two observations. First, sin3 and ume6 mutations are epistatic to rim11 mutations; prior studies have demonstrated their epistasis to ime1 mutations. Second, overexpression of RIM11 can suppress an ime1 missense mutation (ime1-L321F) but not an ime1 deletion. Sequence analysis indicates that RIM11 specifies a protein kinase related to rat glycogen synthase kinase 3 and the Drosophila shaggy/zw3 gene product. Three partially defective rim11 mutations alter residues involved in ATP binding or catalysis, and a completely defective rim11 mutation alters a tyrosine residue that corresponds to the site of an essential phosphorylation for glycogen synthase kinase 3. Immune complexes containing a hemagglutinin (HA) epitope-tagged RIM11 derivative, HA-RIM11, phosphorylate two proteins, p58 and p60, whose biological function is undetermined. In addition, HA-RIM11 immune complexes phosphorylate a functional IME1 derivative but not the corresponding ime1-L321F derivative. We propose that RIM11 stimulates meiotic gene expression through phosphorylation of IME1.

Role of IME1 expression in regulation of meiosis in Saccharomyces cerevisiae.

Two signals are required for meiosis and spore formation in the yeast Saccharomyces cerevisiae: starvation and the MAT products a1 and alpha 2, which determine the a/alpha cell type. These signals lead to increased expression of the IME1 (inducer of meiosis) gene, which is required for sporulation and sporulation-specific gene expression. We report here the sequence of the IME1 gene and the consequences of IME1 expression from the GAL1 promoter. The deduced IME1 product is a 360-amino-acid protein with a tyrosine-rich C-terminal region. Expression of PGAL1-IME1 in vegetative a/alpha cells led to moderate accumulation of four early sporulation-specific transcripts (IME2, SPO11, SPO13, and HOP1); the transcripts accumulated 3- to 10-fold more after starvation. Two sporulation-specific transcripts normally expressed later (SPS1 and SPS2) did not accumulate until PGAL1-IME1 strains were starved, and the intact IME1 gene was not activated by PGAL1-IME1 expression. In a or alpha cells, which lack alpha 2 or a1, expression of PGAL1-IME1 led to the same pattern of IME2 and SPO13 expression as in a/alpha cells, as measured with ime2::lacZ and spo13::lacZ fusions. Thus, in wild-type strains, the increased expression of IME1 in starved a/alpha cells can account entirely for cell type control, but only partially for nutritional control, of early sporulation-specific gene expression. PGAL1-IME1 expression did not cause growing cells to sporulate but permitted efficient sporulation of amino acid-limited cells, which otherwise sporulated poorly. We suggest that IME1 acts primarily as a positive regulator of early sporulation-specific genes and that growth arrest is an independent prerequisite for execution of the sporulation program.

Interaction of yeast repressor-activator protein Ume6p with glycogen synthase kinase 3 homolog Rim11p.

Meiosis and expression of early meiotic genes in the budding yeast Saccharomyces cerevisiae depend upon Rim11p, Ume6p, and Ime1p. Rim11p (also called Mds1p and ScGSK3) is a protein kinase related to glycogen synthase kinase 3 (GSK3); Ume6p is an architectural transcription factor; and Imelp is a Ume6p-binding protein that provides a transcriptional activation domain. Rim11p is required for Ime1p-Ume6p interaction, and prior studies have shown that Rim11p binds to and phosphorylates Ime1p. We show here that Rim11p binds to and phosphorylates Ume6p, as well. Amino acid substitutions in Ume6p that alter a consensus GSK3 site reduce or abolish Rim11p-Ume6p interaction and Rim11p-dependent phosphorylation, and they cause defects in interaction between Ume6p and Ime1p and in meiotic gene expression. Therefore, interaction between Rim11p and Ume6p, resulting in phosphorylation of Ume6p, is required for Ime1p-Ume6p complex formation. Rim11p, like metazoan GSK3beta, phosphorylates both interacting subunits of a target protein complex.

Stimulation of later functions of the yeast meiotic protein kinase Ime2p by the IDS2 gene product.

Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression and down-regulates expression of IME1, which specifies an activator of early meiotic genes that acts independently of Ime2p. We have identified a new gene, IDS2 (for IME2-dependent signaling), which has a functional relationship to Ime2p. An ids2 null mutation delays down-regulation of IME1 and expression of middle and late meiotic genes. In an ime1 null mutant that express IME2 from the GAL1 promoter (ime1 delta PGAL1-IME2 mutant), early meiotic gene expression depends only upon Ime2p. In such strains, Ids2p is dispensable for expression of the early genes HOP1 and SPO13 but is essential for expression of the middle and late genes SPS1, SPS2, and SPS100. Ids2p is also essential for the autoregulatory pathway through which Ime2p activates its own expression via the IME2 upstream activation sequences (UAS). An PGAL1-IME2 derivative that produces a truncated Ime2p (lacking its C-terminal 174 residues) permits IME2 UAS activation in the absence of Ids2p. This observation suggests that Ids2p acts upstream of Ime2p or that Ids2p and Ime2p act in independent, convergent pathways to stimulate IME2 UAS activity. Accumulation of epitope-tagged Ids2p derivatives is greatest in growing cells and declines during meiosis. We propose that Ids2p acts indirectly to modify Ime2p activity, thus permitting Ime2p to carry out later meiotic functions.