Article

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.

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    • "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). "
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    • "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. "
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