Abstract #46

Association of pH-sensing Machinery with Endosomal Compartments. Jacob Boysen^1,2, Aaron Mitchell^1,2. 1) Department of Microbiology, Columbia University, New York, NY; 2) Integrated Program in Cellular, Molecular and Biophysical Studies, Columbia University, New York, NY.
   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. Evidence for the participation of the seven-transmembrane domain protein Dfg16 in this process will be presented. Our findings raise the possibility that Bro1-domain proteins may be differentially regulated in the same cell, thereby coupling endosome metabolism to signaling.

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