Bro1-domain proteins such as yeast Bro1 and mammalian AIP1/Alix are well-established participants in endosome metabolism. The Bro1-domain interacts with endosomal surface protein Snf7/Vps32 in yeast, a subunit of the ESCRT complex. Yeast Bro1-domain protein Rim20 has no role in endosome function, but is required for alkaline pH-stimulated cleavage of transcription factor Rim101. Rim20-GFP is cytoplasmic under acidic conditions but concentrated in punctate foci under alkaline conditions. Bro1-GFP also accumulates in foci, but they are more numerous under acidic than alkaline conditions. Colocalization experiments indicate that some Rim20-GFP foci correspond to Bro1-RFP foci, whereas others do not. Rim8, Rim9, Rim21, Dfg16, Snf7, Vps20, Vps23, and Vps25, which are required for Rim101 cleavage, are required for appearance of Rim20-GFP foci. ESCRT complexes accumulate on endosome-derived compartments in cells that lack the AAA-ATPase Vps4. We find that Rim20-GFP foci accumulate in a vps4 mutant background independently of external pH, Rim101 pathway-specific genes, and most ESCRT subunit genes except for SNF7. Rim20-GFP foci seem to represent endosomes, because they colocalize with Snf7-RFP and because they correspond to a perivacuolar compartment in the vps4 strain. We propose that alkaline growth conditions alter the endosomal surface to favor Rim20-Snf7 interaction and Rim101 cleavage. Our findings raise the possibility that Bro1-domain proteins may be differentially regulated in the same cell, thereby coupling endosome metabolism to signaling.
INTRODUCTIONAll cells recognize and respond to external signals. For microorganisms, the responses are vital for adaptation to a changeable environment. For multicellular organisms, the responses are critical for coordination of developmental and physiological processes. The plasma membrane is well recognized as the chief sensory organelle of the cell, where receptors undergo environmentally directed changes that relay information to the cytoplasm and nucleus. The endocytic machinery is routinely used for down-regulation of receptors and metabolism or destruction of ligands. 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.Rim20 is required for proteolytic activation of the transcription factor Rim101 . Rim101 cleavage removes a ϳ100-residue C-terminal PEST-like region, allowing the N-terminal zinc finger region to effect transcriptional changes (Li and Mitchell, 1997;Lamb et al., 2001). Ultimately, cleaved Rim101 is required for expression of many alkaline pH-inducible genes (Lamb et al., 2001...