Constitutive Activation of the pH-Responsive Rim101 Pathway in Yeast Mutants Defective in Late Steps of the MVB/ESCRT Pathway

Abstract

In many fungi, transcriptional responses to alkaline pH are mediated by conserved signal transduction machinery. In the homologous system in Saccharomyces cerevisiae, the zinc-finger transcription factor Rim101 is activated under alkaline conditions to regulate transcription of target genes. The activation of Rim101 is exerted through proteolytic processing of its C-terminal inhibitory domain. Regulated processing of Rim101 requires several proteins, including the calpain-like protease Rim13/Cpl1, a putative protease scaffold Rim20, putative transmembrane proteins Rim9, and Rim21/Pal2, and Rim8/Pal3 of unknown biochemical function. To identify new regulatory components and thereby determine the order of action among the components in the pathway, we screened for suppressors of rim9⌬ and rim21⌬ mutations. Three identified suppressors-did4/ vps2, vps24, and vps4-all belonged to "class E" vps mutants, which are commonly defective in multivesicular body sorting. These mutations suppress rim8, rim9, and rim21 but not rim13 or rim20, indicating that Rim8, Rim9, and Rim21 act upstream of Rim13 and Rim20 in the pathway. Disruption of DID4, VPS24, or VPS4, by itself, uncouples pH sensing from Rim101 processing, leading to constitutive Rim101 activation. Based on extensive epistasis analysis between pathway-activating and -inactivating mutations, a model for architecture and regulation of the Rim101 pathway is proposed.

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

“…Third, we show here that the functions of Dfg16, Rim8, Rim9, and Rim21 may be bypassed by a vps4 defect, whereas the function of Rim13 cannot. A recent independent study reported completely convergent findings (Hayashi et al, 2005). Finally, the fact that Rim13 and Rim101 are not required for Rim20 focus formation is consistent with the proposal that Rim20 functions by recruiting both Rim13 and Rim101, if one assumes that a Rim20 focus serves as a recruitment site.…”

“…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).…”

“…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.…”

The Rim101 Pathway Is Involved in Rsb1 Expression Induced by Altered Lipid Asymmetry

“…In the pH-responsive pathway, Rim13, Rim20, and Rim21 are all located upstream of the transcription factor Rim101 (Hayashi et al, 2005;Boysen and Mitchell, 2006). Rim21 and Dfg16, both multimembrane-spanning proteins, and their homologues are thought to serve as pH sensors (Peñ alva and Arst, 2004; Barwell et al, 2005;Herranz et al, 2005;Boysen and Mitchell, 2006).…”

“…The pFI1 plasmid (Hayashi et al, 2005), which encodes N-terminally triple HA-tagged Rim101 (HA-Rim101), was a kind gift from Dr. Tatsuya Maeda (Tokyo University, Japan). The pIKD412 plasmid, which is derived from the pRS423 vector (2 , HIS3 marker) (Christianson et al, 1992), encodes RSB1-HA under the control of the RSB1 promoter (P RSB1 ).…”