Role of the leader sequence in tobacco pectin methylesterase secretion |
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Authors: | Dorokhov Yuri L Skurat Eugene V Frolova Olga Yu Gasanova Tatjana V Ivanov Peter A Ravin Nikolay V Skryabin Konstantin G Mäkinen Kristiina M Klimyuk Viktor I Gleba Yuri Yu Atabekov Joseph G |
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Affiliation: | Department of Virology, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninsky Gory 1, Laboratory Building A, Moscow 119992, Russia. |
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Abstract: | We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting. |
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Keywords: | aa amino acid proPME, full-length PME gene including the 5′-sequence coding for the N-terminal PME pro-sequence region as-proPME, proPME gene in antisense orientation BFA, brefeldin A CW, cell wall ER, endoplasmic reticulum GFP, green fluorescent protein MP, movement protein PME, mature, processed pectin methylesterase PME(395A396A), PME mutant with Ala substitutions in its active center PS, N-terminal pro-sequence SS, spacer sequence TM, transmembrane |
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