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
Chromochloris zofingiensis represents an industrially relevant and unique green alga,given its capability of synthesizing triacylglycerol(TAG)and astaxanthin simultaneously for storage in lipid droplets(LDs).To furthe...Chromochloris zofingiensis represents an industrially relevant and unique green alga,given its capability of synthesizing triacylglycerol(TAG)and astaxanthin simultaneously for storage in lipid droplets(LDs).To further decipher lipid metabolism,the nitrogen deprivation(ND)-induced LDs from C.zofingiensis were isolated,purified,and subjected to proteomic analysis.Intriguingly,many C.zofingiensis LD proteins had no orthologs present in LD proteome of the model alga Chlamydomonas reinhardtii.Seven novel LD proteins(i.e.,two functionally unknown proteins,two caleosins,two lipases,and one L-gulonolactone oxidase)and the major LD protein(MLDP),which were all transcriptionally up-regulated by ND,were selected for further investigation.Heterologous expression in yeast demonstrated that all tested LD proteins were localized to LDs and all except the two functionally unknown proteins enabled yeast to produce more TAG.MLDP could restore the phenotype of mldp mutant strain and enhance TAG synthesis in wildtype strain of C.reinhardtii.Although MLDP and caleosins had a comparable abundance in LDs,they responded distinctly to ND at the transcriptional level.The two lipases,instead of functioning as TAG lipases,likely recycled polar lipids to support TAG synthesis.For the first time,we reported that L-gulonolactone oxidase was abundant in LDs and facilitated TAG accumulation.Moreover,we also proposed a novel working model for C.zofingiensis LDs.Taken together,our work unravels the unique characteristics of C.zofingiensis LDs and provides insights into algal LD biogenesis and TAG synthesis,which would facilitate genetic engineering of this alga for TAG improvement.展开更多
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.
基金partially funded by grants from the National Youth Thousand Talents Program of China,Double-First Class Initiative of Peking University,Chinathe China Postdoctoral Science Foundation(Grant No.2018M641070)
文摘Chromochloris zofingiensis represents an industrially relevant and unique green alga,given its capability of synthesizing triacylglycerol(TAG)and astaxanthin simultaneously for storage in lipid droplets(LDs).To further decipher lipid metabolism,the nitrogen deprivation(ND)-induced LDs from C.zofingiensis were isolated,purified,and subjected to proteomic analysis.Intriguingly,many C.zofingiensis LD proteins had no orthologs present in LD proteome of the model alga Chlamydomonas reinhardtii.Seven novel LD proteins(i.e.,two functionally unknown proteins,two caleosins,two lipases,and one L-gulonolactone oxidase)and the major LD protein(MLDP),which were all transcriptionally up-regulated by ND,were selected for further investigation.Heterologous expression in yeast demonstrated that all tested LD proteins were localized to LDs and all except the two functionally unknown proteins enabled yeast to produce more TAG.MLDP could restore the phenotype of mldp mutant strain and enhance TAG synthesis in wildtype strain of C.reinhardtii.Although MLDP and caleosins had a comparable abundance in LDs,they responded distinctly to ND at the transcriptional level.The two lipases,instead of functioning as TAG lipases,likely recycled polar lipids to support TAG synthesis.For the first time,we reported that L-gulonolactone oxidase was abundant in LDs and facilitated TAG accumulation.Moreover,we also proposed a novel working model for C.zofingiensis LDs.Taken together,our work unravels the unique characteristics of C.zofingiensis LDs and provides insights into algal LD biogenesis and TAG synthesis,which would facilitate genetic engineering of this alga for TAG improvement.
