Subcellular localization and membrane topology of the melon ethylene receptor CmERS1.
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 (Cucumis melo) ethylene receptor that has three putative transmembrane domains at the N terminus. Analyses using membrane fractionation and green fluorescent protein 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 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 transmembrane 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.
Ma
Biao
B
Gene Research Center, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan.
Cui
Min-Long
ML
Sun
Hyeon-Jin
HJ
Takada
Keita
K
Mori
Hitoshi
H
Kamada
Hiroshi
H
Ezura
Hiroshi
H
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D019662:Cucurbitaceae; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*
D010940:Plant Proteins; D011956:Receptors, Cell Surface; C083750:ethylene receptors, plant