Intervention of Bro1 in pH-Responsive Rim20 Localization in Saccharomyces cerevisiae

Department of Microbiology, Columbia University, New York, New York 10032, USA.
Eukaryotic Cell (Impact Factor: 3.18). 02/2010; 9(4):532-8. DOI: 10.1128/EC.00027-10
Source: PubMed

ABSTRACT Yeast cells contain two Bro1 domain proteins: Bro1, which is required for endosomal trafficking, and Rim20, which is required for the response to the external pH via the Rim101 pathway. Rim20 associates with endosomal structures under alkaline growth conditions, when it promotes activation of Rim101 through proteolytic cleavage. We report here that the pH-dependent localization of Rim20 is contingent on the amount of Bro1 in the cell. Cells that lack Bro1 have increased endosomal Rim20-green fluorescent protein (GFP) under acidic conditions; cells that overexpress Bro1 have reduced endosomal Rim20-GFP under acidic or alkaline conditions. The novel endosomal association of Rim20-GFP in the absence of Bro1 requires ESCRT components including Vps27 but not specific Rim101 pathway components such as Dfg16. Vps27 influences the localization of Bro1 but is not required for RIM101 pathway activation in wild-type cells, thus suggesting that Rim20 enters the Bro1 localization pathway when a vacancy exists. Despite altered localization of Rim20, the lack of Bro1 does not bypass the need for signaling protein Dfg16 to activate Rim101, as evidenced by the expression levels of the Rim101 target genes RIM8 and SMP1. Therefore, endosomal association of Rim20 is not sufficient to promote Rim101 activation.

  • Source
    • "Thus, the current view is that PalA links PacC to Vps32-containing complexes, ‘presenting’ the PacC substrate to the signalling protease PalB (Rim20p), itself an ESCRT-III interactor (Peñas et al., 2007; Rodríguez-Galán et al., 2009; Xu and Mitchell, 2001). Our data strongly indicate that signalling necessitates the Vps32-dependent PalA recruitment to the plasma membrane [in agreement with the finding that endosomal association of the PalA orthologue Rim20p is not sufficient to promote pH signalling (Boysen et al., 2010)]. They are consistent with the model in Fig. 9 in which most if not all these ‘late’ reactions would take place within ESCRT-containing pH signalling complexes associated with the plasma membrane, rather than endosomes as previously suggested (Mitchell, 2008; Peñalva et al., 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The fungal pal/RIM signalling pathway, which regulates gene expression in response to environmental pH involves, in addition to dedicated proteins, several components of ESCRT complexes, which suggested that pH signalling proteins assemble on endosomal platforms. In Aspergillus nidulans, dedicated Pal proteins include the plasma membrane receptor PalH and its coupled arrestin, PalF, which becomes ubiquitylated in alkaline pH conditions, and three potentially endosomal ESCRT-III associates, including Vps32 interactors PalA and PalC and Vps24 interactor calpain-like PalB. We studied the subcellular locations at which signalling takes place after activating the pathway by shifting ambient pH to alkalinity. Rather than localising to endosomes, Vps32 interactors PalA and PalC transiently colocalise at alkaline-pH-induced cortical structures in a PalH-, Vps23- and Vps32-dependent but Vps27-independent manner. These cortical structures are much more stable when Vps4 is deficient, indicating that their half-life depends on ESCRT-III disassembly. Pull-down studies revealed that Vps23 interacts strongly with PalF, but co-immunoprecipitates exclusively with ubiquitylated PalF forms from extracts. We demonstrate that Vps23-GFP, expressed at physiological levels, is also recruited to cortical structures, very conspicuous in vps27Δ cells in which the prominent signal of Vps23-GFP on endosomes is eliminated, in a PalF- and alkaline pH-dependent manner. Dual-channel epifluorescence microscopy showed that PalC arrives at cortical complexes before PalA. As PalC recruitment is PalA independent and PalA recruitment is PalC dependent but PalB independent, these data complete the participation order of Pal proteins in the pathway and strongly support a model in which pH signalling takes place in ESCRT-containing, plasma-membrane-associated, rather than endosome-associated, complexes.
    Journal of Cell Science 02/2012; 125(Pt 7):1784-95. DOI:10.1242/jcs.098897 · 5.33 Impact Factor
    • "Studies pioneered by the Mitchell laboratory (Carnegie Mellon University, Pittsburgh, PA) established involvement of all components of ESCRT-I and ESCRT-II, and Vps20 and Vps32 of ESCRT-III, in the proteolytic activation of the yeast PacC orthologue Rim101p (Boysen et al., 2010; Boysen and Mitchell, 2006; Hayashi et al., 2005; Rothfels et al., 2005; Xu et al., 2004). Our pH shift experiments established unambiguously that the absence of Vps23, Vps36, Vps20 or Vps32 prevents PacC processing. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Aspergillus pal pathway hijacks ESCRT proteins into ambient pH signalling complexes. We show that components of ESCRT-0, ESCRT-I, ESCRT-II and ESCRT-III are nearly essential for growth, precluding assessment of null mutants for pH signalling or trafficking. This severely debilitating effect is rescued by loss-of-function mutations in two cation tolerance genes, one of which, sltA, encodes a transcription factor whose inactivation promotes hypervacuolation. Exploiting a conditional expression sltA allele, we demonstrate that deletion of vps27 (ESCRT-0), vps23 (ESCRT-I), vps36 (ESCRT-II), or vps20 or vps32 (both ESCRT-III) leads to numerous small vacuoles, a phenotype also suppressed by SltA downregulation. This situation contrasts with normal vacuoles and vacuole-associated class E compartments seen in Saccharomyces cerevisiae ESCRT null mutants. Exploiting the suppressor phenotype of sltA(-) mutations, we establish that Vps23, Vps36, Vps20 and Vps32 are essential for pH signalling. Phosphatidylinositol 3-phosphate-recognising protein Vps27 (ESCRT-0) is not, consistent with normal pH signalling in rabB null mutants unable to recruit Vps34 kinase to early endosomes. In contrast to the lack of pH signalling in the absence of Vps20 or Vps32, detectable signalling occurs in the absence of ESCRT-III subunit Vps24. Our data support a model in which certain ESCRT proteins are recruited to the plasma membrane to mediate pH signalling.
    Journal of Cell Science 12/2011; 124(Pt 23):4064-76. DOI:10.1242/jcs.088344 · 5.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The four protein complexes termed endosomal sorting complexes required for transport (ESCRT) are key mediators of multivesicular body sorting/formation, retroviral budding and cell abscission, which share a membrane deformation process with the same topological change: vesicles budding away from the cytoplasm. Independent studies of the signal transduction pathways that mediate ambient pH sensing and adaptation in yeast and fungi revealed that these pathways share a conserved signaling mechanism that utilizes ESCRT complexes for its activation. This pathway in Saccharomyces cerevisiae, termed the Rim101 pathway, consists of both a sensing complex, which senses ambient alkaline pH, and a proteolytic complex, which proteolyzes and thereby activates the key transcription factor Rim101. Since the proteolytic complex is thought to be formed and activated on a platform of a multimerized ESCRT-III component Snf7, the organization, regulation and function of this pathway are dependent on the function of ESCRT complexes.
    FEBS Journal 02/2012; 279(8):1407-13. DOI:10.1111/j.1742-4658.2012.08548.x · 3.99 Impact Factor
Show more


Available from