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

Abstract

In yeast, external alkalization and alteration in plasma membrane lipid asymmetry are sensed by the Rim101 pathway. It is currently under debate whether the signal elicited by external alkalization is transduced to downstream molecules at the plasma membrane or via endocytosis of the Rim21 sensor protein at the late endosome. We found that the downstream molecules, including arrestin-related protein Rim8, calpain-like protein Rim13, and scaffold protein Rim20, accumulated at the plasma membrane upon external alkalization and that the accumulation was dependent on Rim21. Snf7, an endosomal sorting complex required for transport (ESCRT) III subunit also essential for the Rim101 pathway, localized to the plasma membrane, in addition to the late endosome, under alkaline conditions. Snf7 at the plasma membrane 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 impaired. Considering this information as a whole, we propose that Rim101 signaling proceeds at the plasma membrane. We also found that activity of the Rsp5 ubiquitin ligase was required for recruiting the downstream molecules to the plasma membrane, suggesting that ubiquitination mediates Rim101 signaling at the plasma membrane.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00408-14.

ACKNOWLEDGMENTS

We thank T. Maeda (Institute of Molecular and Cellular Biosciences, University of Tokyo) for providing the HA-Rim101 plasmid (pFI1), H. Nakatogawa (Frontier Research Center, Tokyo Institute of Technology) for the template vector for N-terminal tagging of the AID tag (pCT1), and F. Abe (College of Science and Engineering, Aoyama Gakuin University) for the rsp5-1 strain. The template plasmid for the AID tag was provided by the National Bio-Resource Project of the MEXT, Japan. We are grateful to E. A. Sweeney and T. Toyokuni for scientific editing of the manuscript.

This work was supported by a Grant-in-Aid for Scientific Research (C) (25440038) and a Grant-in-Aid for Young Scientists (B) (23770135) to K.O. and a Grant-in-Aid for Challenging Exploratory Research (25650059) to A.K. from the Japan Society for the Promotion of Science. This work was also supported by the Naito Foundation Subsidy for Promotion of Specific Research Projects to K.O. from the Naito Foundation.