Signaling Events of the Rim101 Pathway Occur at the PlasmaMembrane in a Ubiquitination-Dependent Manner

In yeast, external alkalization and alteration in plasmamembrane lipid asymmetry are sensed by the Rim101 pathway. It is cur-rently under debate whether the signal elicited by external alkalization is transduced to downstreammolecules at the plasma membrane or via endocytosis of the Rim21 sensor protein at the late endosome.We found that the downstreammolecules, in-cluding arrestin-related protein Rim8, calpain-like protein Rim13, and scaffold protein Rim20, accumulated at the plasmamem-brane upon external alkalization and that the accumulation was dependent on Rim21. Snf7, an endosomal sorting complex re-quired for transport (ESCRT) III subunit also essential for the Rim101 pathway, localized to the plasmamembrane, in addition to the late endosome, under alkaline conditions. Snf7 at the plasmamembrane but not at the late endosome was shown to be involved in Rim101 signaling. In addition, the Rim101 pathway was normally activated, even when endocytosis was severely im-paired. Considering this information as a whole, we propose that Rim101 signaling proceeds at the plasmamembrane. We also found that activity of the Rsp5 ubiquitin ligase was required for recruiting the downstreammolecules to the plasmamembrane, suggesting that ubiquitinationmediates Rim101 signaling at the plasmamembrane. Adaptation to external pH is vital for natural growth and for thepathogenicity of certain microorganisms, so understanding themechanisms for such adaptation is important in biology,med-icine, and agriculture. In the filamentous fungus Aspergillus nidu-lans and the budding yeast Saccharomyces cerevisiae, alkalizatio