Plant sensitive factor attachment protein receptors （SNAREs） encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the app...Plant sensitive factor attachment protein receptors （SNAREs） encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the application of innovative experimental approaches highlighted that members of the same gene sub-family often have different functional specificities. In this work, two closely related Qc-SNAREs--the AtSYP51 and the AtSYP52--are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs （syntaxin of plants） act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs （i-SNAREs） when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.展开更多
Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed i...Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets （LD）, whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum （ER）. Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin- green fluorescent protein （GFP） peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein （BiP）, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.展开更多
文摘Plant sensitive factor attachment protein receptors （SNAREs） encoded by genes of the same sub-family are generally considered as redundant in promoting vesicle-associated membrane fusion events. Nonetheless, the application of innovative experimental approaches highlighted that members of the same gene sub-family often have different functional specificities. In this work, two closely related Qc-SNAREs--the AtSYP51 and the AtSYP52--are compared in their ability to influence different secretory pathways. Their role in the vesicle sorting to the central vacuole has been revised and they were found to have a novel inhibitory function. When transiently overexpressed, the SYP51 and the SYP52 distributed between the TGN and the tonoplast. Our data demonstrate that these SYPs （syntaxin of plants） act as t-SNARE when present on the membrane of TGN/PVC, whereas they behave as inhibitory or interfering SNAREs （i-SNAREs） when they accumulate on the tonoplast. Moreover, the performed functional analysis indicated that the AtSYP51 and the AtSYP52 roles differ in the traffic to the vacuole. The findings are a novel contribution to the functional characterization of plant SNAREs that reveals additional non-fusogenic roles.
文摘Oleosin, caleosin and steroleosin are normally expressed in developing seed cells and are targeted to oil bodies. In the present work, the cDNA of each gene tagged with fluorescent proteins was transiently expressed into tobacco protoplasts and the fluorescent patterns observed by confocal laser scanning microscopy. Our results indicated clear differences in the endocellular localization of the three proteins. Oleosin and caleosin both share a common structure consisting of a central hydrophobic domain flanked by two hydrophilic domains and were correctly targeted to lipid droplets （LD）, whereas steroleosin, characterized by an N-terminal oil body anchoring domain, was mainly retained in the endoplasmic reticulum （ER）. Protoplast fractionation on sucrose gradients indicated that both oleosin and caleosin- green fluorescent protein （GFP） peaked at different fractions than where steroleosin-GFP or the ER marker binding immunoglobulin protein （BiP）, were recovered. Chemical analysis confirmed the presence of triacylglycerols in one of the fractions where oleosin-GFP was recovered. Finally, only oleosin- and caleosin-GFP were able to reconstitute artificial oil bodies in the presence of triacylglycerols and phospholipids. Taken together, our results pointed out for the first time that leaf LDs can be separated by the ER and both oleosin or caleosin are selectively targeted due to the existence of selective mechanisms controlling protein association with these organelles.
