Characterization of the topology and functional domains of RKTG.
RKTG (Raf kinase trapping to Golgi) is exclusively localized at the Golgi apparatus and functions as a spatial regulator of Raf-1 kinase by sequestrating Raf-1 to the Golgi. Based on the structural similarity with adiponectin receptors, RKTG was predicted to be a seven-transmembrane protein with a cytosolic N-terminus, distinct from classical GPCRs (G-protein-coupled receptors). We analysed in detail the topology and functional domains of RKTG in this study. We determined that the N-terminus of RKTG is localized on the cytosolic side. Two short stretches of amino acid sequences at the membrane proximal to the N- and C-termini (amino acids 61-71 and 299-303 respectively) were indispensable for Golgi localization of RKTG, but were not required for the interaction with Raf-1. The three loops facing the cytosol between the transmembrane domains had different roles in Golgi localization and Raf-1 interaction. While the first cytosolic loop was only important for Golgi localization, the third cytosolic loop was necessary for both Golgi localization and Raf-1 sequestration. Taken together, these findings suggest that RKTG is a type III membrane protein with its N-terminus facing the cytosol and multiple sequences are responsible for its localization at the Golgi apparatus and Raf-1 interaction. As RKTG is the first discovered Golgi protein with seven transmembrane domains, the knowledge derived from this study would not only provide structural information about the protein, but also pave the way for future characterization of the unique functions of RKTG in the regulation of cell signalling.
Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, 294 Taiyuan Road, Shanghai 200031, China.
Xiaolin
X
Luo
Lin
L
Feng
Xiaomeng
X
Jiang
Fei
F
Xiao
Zhenzhen
Z
Wang
Gen-Sheng
GS
Feng
Yan
Y
Chen
D002460:Cell Line; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C532354:PAQR3 protein, human; D019908:Proto-Oncogene Proteins c-raf