Plant Physiology Preview Published on April 14, 2006; 10.1104/pp.106.080523
Received March 15, 2006
Returned for revision April 9, 2006
Accepted April 9, 2006
Subcellular Localization and Membrane Topology of the Melon Ethylene Receptor CmERS1
Biao Ma , Min-Long Cui , Hyeon-Jin Sun , Keita Takada , Hitoshi Mori , Hiroshi Kamada , and Hiroshi Ezura *
Gene Research Center, University of Tsukuba, Tennodai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho Chikusa-ku, Nagoya 464-8601, Japan
* Corresponding author; email: ezura{at}gene.tsukuba.ac.jp.
Ethylene receptors are multispanning membrane proteins that negatively regulate ethylene responses via the formation of a signaling complex with downstream elements. To better understand their biochemical functions, we investigated the membrane topology and subcellular localization of CmERS1, a melon ethylene receptor that has three putative transmembrane (TM) domains at the N-terminus. Analyses using membrane fractionation and GFP imaging approaches indicate that CmERS1 is predominantly associated with the endoplasmic reticulum (ER) membrane. Detergent treatments of melon microsomes showed that the receptor protein is integrally bound to the ER membrane. A protease protection assay and N-glycosylation analysis were used to determine the membrane topology. The results indicate that CmERS1 spans the membrane three times, with its N-terminus facing the luminal space and the large C-terminal portion lying on the cytosolic side of the ER membrane. This orientation provides a platform for interaction with the cytosolic signaling elements. The three N-terminal TM segments were found to function as topogenic sequences to determine the final topology. High conservation of these topogenic sequences in all ethylene receptor homologs identified thus far suggests that these proteins may share the same membrane topology.
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