Components of the ESCRT Pathway, DFG16, and YGR122w Are Required for Rim101 To Act as a Corepressor with Nrg1 at the Negative Regulatory Element of the DIT1 Gene of Saccharomyces cerevisiae

Abstract

The divergently transcribed DIT1 and DIT2 genes of Saccharomyces cerevisiae, which belong to the mid-late class of sporulation-specific genes, are subject to Ssn6-Tup1-mediated repression in mitotic cells. The Ssn6-Tup1 complex, which is required for repression of diverse sets of coordinately regulated genes, is known to be recruited to target genes by promoter-specific DNA-binding proteins. In this study, we show that a 42-bp negative regulatory element (NRE) present in the DIT1-DIT2 intergenic region consists of two distinct subsites and that a multimer of each subsite supports efficient Ssn6-Tup1-dependent repression of a CYC1-lacZ reporter gene. By genetic screening procedures, we identified DFG16, YGR122w, VPS36, and the DNA-binding proteins Rim101 and Nrg1 as potential mediators of NRE-directed repression. We show that Nrg1 and Rim101 bind simultaneously to adjacent target sites within the NRE in vitro and act as corepressors in vivo. We have found that the ability of Rim101 to be proteolytically processed to its active form and mediate NRE-directed repression not only depends on the previously characterized RIM signaling pathway but also requires Dfg16, Ygr122w, and components of the ESCRT trafficking pathway. Interestingly, Rim101 was processed in bro1 and doa4 strains but was unable to mediate efficient repression.The yeast Saccharomyces cerevisiae can respond to changes in nutrient availability and environmental conditions by switching from one growth form to another. Extracellular cues are interpreted by various signal transduction pathways that act by both independent and convergent mechanisms to coordinate morphogenetic events. The different regulatory cascades and the interplay between them ultimately modulate the activity of target transcription factors and progression through the cell cycle. Haploid cells that are starved for glucose and diploid cells that are starved for nitrogen switch from growth as single budding cells to growth as long filaments of connected cells, with haploid cells becoming particularly adept at invasive growth and penetration into solid agar medium (reviewed in reference 63). Starvation of a diploid MATa/MAT␣ cell for an essential nutrient in the absence of glucose and in the presence of a nonfermentable carbon source directs the cell into the pathway for spore formation (reviewed in reference 40). The nutritional signaling leads to arrest of the starved cell at G 1 and contributes in a diploid to the expression of two key regulatory genes, IME1 and IME2, which encode a transcriptional activator and a protein kinase, respectively (reviewed in reference 43). These regulators set in motion the sporulation program that consists of meiotic DNA replication, recombination, the two meiotic divisions, and encapsulation of each haploid nucleus within a multilayered spore wall (reviewed in reference 48). This coordinated series of genetic and morphological events depends on the sequential expression of temporally distinct classes of sporulation-specific genes (18, 68).The regulated...

Control of Bro1-Domain Protein Rim20 Localization by External pH, ESCRT Machinery, and theSaccharomyces cerevisiaeRim101 Pathway

“…On the other hand, mutants defective in Vps4 or in Vps2 and Vps24, the ESCRT-III subunits that recruit Vps4, were capable of producing cleaved Rim101 (Xu et al, 2004). Largely consistent results have been obtained independently for S. cerevisiae (Barwell et al, 2005;Hayashi et al, 2005;Rothfels et al, 2005) and other fungi (Kullas et al, 2004;Xu et al, 2004;Cornet et al, 2005). These findings led to the proposal that vesicle-associated Snf7 is required for productive interaction with Rim20, leading to Rim101 cleavage (Xu et al, 2004).…”

“…To test this hypothesis, we monitored processing of a Ura3-V5-Rim101 fusion protein in a panel of VPS4 and vps4 strains. In the VPS4 strains, we verified that Ura3-V5-Rim101 has the same requirements described previously for native Rim101 ( Figure 6A; Xu et al, 2004;Barwell et al, 2005;Rothfels et al, 2005). These requirements include most ES-CRT-I, -II, and -III subunits and all Rim101 pathway proteins except Rim101 itself.…”

“…Rim9, Rim21, and Dfg16 are the three membrane proteins in the Rim101 pathway, and Rim8 probably associates with Dfg16 or Rim21, based on analysis of its Aspergillus homolog (Penalva and Arst, 2002;Barwell et al, 2005;Herranz et al, 2005). Vps20, Vps23, and Vps25 are ESCRT complex subunits that are required for Rim101 cleavage (Xu et al, 2004;Hayashi et al, 2005;Rothfels et al, 2005). The other class, which included rim13 and rim101 mutants, had a slightly increased frequency of foci under alkaline conditions (p Ͻ 0.05), an issue taken up in the Discussion.…”

“…However, there is growing evidence that endocytosis plays diverse roles in signal transduction and cell physiology (Di Fiore and De Camilli, 2001;Conner and Schmid, 2003). Recent analysis of the fungal Rim101 pH-response pathway suggests that the endocytic machinery may participate directly in signaling (Kullas et al, 2004;Xu et al, 2004;Barwell et al, 2005;Rothfels et al, 2005). Our focus here is the interaction of endosomal multivesicular body (MVB) formation components with Rim20, a key participant in pH-dependent signaling.…”

“…The PacC processing regulators have proven to be broadly conserved among fungi (Penalva and Arst, 2002). The S. cerevisiae homologues, which are required along with ESCRT subunits for Rim101 cleavage, include the arrestin homolog Rim8, the transmembrane proteins Dfg16, Rim21, and Rim9, as well as the scaffold Rim20 and the protease Rim13 (Futai et al, 1999;Penalva and Arst, 2002;Barwell et al, 2005;Rothfels et al, 2005). The transmembrane proteins have generally been thought to serve as sensors of external pH (Penalva and Arst, 2002).…”