Apicomplexan encompass a variety of intracellular parasites responsible for significant diseases in humans and animals,such as malaria and toxoplasmosis.Among these,Toxoplasma gondii(T.gondii)serves as an exemplary or...Apicomplexan encompass a variety of intracellular parasites responsible for significant diseases in humans and animals,such as malaria and toxoplasmosis.Among these,Toxoplasma gondii(T.gondii)serves as an exemplary organism for understanding the intricate biological characteristics of the phylum.At the parasite’s apical tip,a striated fiber was discovered and proposed to be evotionarily derived from the algal flagellum.However,the protein composition(the striated fiber assemblin,SFA)of this fiber remains poorly understood.Here,we took advantage of a proximity biotin labeling approach to identify potential SFA proteins by fusion of an engeneered biotin ligase TurboID with SFA2,from which we identified several novel components of the fiber.Evolutionary analysis suggested that SFA4 is conserved in the Apicomplexa phylum with the exception of piroplasmida,while SFA5 is specific to species of Sarcocystidae.Confocal imaging analysis showed that SFA4 and SFA5 are substantially co-localized with the bait SFA2.Using a new version(OsTIR1^(F74G))of the plant auxin-inducible degron system that we adapted in this study,we found that SFA4 and SFA5 were efficiently depleted by addition of a much lower concentration of inducer(5-Ph-IAA,instead of IAA).Detailed phenotypical analyses demonstrated that SFA5 is essential for daughter parasite formation and separation of parasite nuclei during division,supporting the localization of SFA5 at the striated fiber that connects the centriole and the apical conoid.The mouse assay suggested the essentiality of SFA5 in the parasites.Thus,this study provided a new repertoile of the stiated fiber,and new data supporting association of the fiber with parasite division.展开更多
Nascent pectin and glucuronoxylan were prepared from membrane-bound enzymes obtained from pea epicotyls. They had previously been shown to exhibit a protein- and pH-dependent pattern of binding to cell wall ghosts and...Nascent pectin and glucuronoxylan were prepared from membrane-bound enzymes obtained from pea epicotyls. They had previously been shown to exhibit a protein- and pH-dependent pattern of binding to cell wall ghosts and to xy-loglucan extracted from cell walls prepared from pea epicotyls;maximum binding required a pH of 3-4, and the pres-ence of cell wall proteins, namely assemblins. To determine whether wall polysaccharides deposited in cell walls be-have in the same manner as nascent polymers, radioactively labeled EDTA-soluble polymers were prepared from newly-deposited pea epicotyl cell walls. Different enzyme treatments followed by column chromatography, in addition to complete acid hydrolysis followed by paper and thin layer chromatography, indicated the presence of pectin, to-gether with smaller amounts of glucuronoxylan, in this EDTA-soluble extract. These radioactively labeled polysaccha-rides were found to bind to cell wall ghosts and to xyloglucan extracted from the second and third internodes of pea epicotyls cell walls in a pH-dependent manner, similar to the binding pattern obtained with nascent polymers. Maxi-mum binding occurred at pH 3-4, and also required the presence of protein.展开更多
基金funded by the National Key Research and Development Project of China(Grant Nos.2022YFD1800200).
文摘Apicomplexan encompass a variety of intracellular parasites responsible for significant diseases in humans and animals,such as malaria and toxoplasmosis.Among these,Toxoplasma gondii(T.gondii)serves as an exemplary organism for understanding the intricate biological characteristics of the phylum.At the parasite’s apical tip,a striated fiber was discovered and proposed to be evotionarily derived from the algal flagellum.However,the protein composition(the striated fiber assemblin,SFA)of this fiber remains poorly understood.Here,we took advantage of a proximity biotin labeling approach to identify potential SFA proteins by fusion of an engeneered biotin ligase TurboID with SFA2,from which we identified several novel components of the fiber.Evolutionary analysis suggested that SFA4 is conserved in the Apicomplexa phylum with the exception of piroplasmida,while SFA5 is specific to species of Sarcocystidae.Confocal imaging analysis showed that SFA4 and SFA5 are substantially co-localized with the bait SFA2.Using a new version(OsTIR1^(F74G))of the plant auxin-inducible degron system that we adapted in this study,we found that SFA4 and SFA5 were efficiently depleted by addition of a much lower concentration of inducer(5-Ph-IAA,instead of IAA).Detailed phenotypical analyses demonstrated that SFA5 is essential for daughter parasite formation and separation of parasite nuclei during division,supporting the localization of SFA5 at the striated fiber that connects the centriole and the apical conoid.The mouse assay suggested the essentiality of SFA5 in the parasites.Thus,this study provided a new repertoile of the stiated fiber,and new data supporting association of the fiber with parasite division.
文摘Nascent pectin and glucuronoxylan were prepared from membrane-bound enzymes obtained from pea epicotyls. They had previously been shown to exhibit a protein- and pH-dependent pattern of binding to cell wall ghosts and to xy-loglucan extracted from cell walls prepared from pea epicotyls;maximum binding required a pH of 3-4, and the pres-ence of cell wall proteins, namely assemblins. To determine whether wall polysaccharides deposited in cell walls be-have in the same manner as nascent polymers, radioactively labeled EDTA-soluble polymers were prepared from newly-deposited pea epicotyl cell walls. Different enzyme treatments followed by column chromatography, in addition to complete acid hydrolysis followed by paper and thin layer chromatography, indicated the presence of pectin, to-gether with smaller amounts of glucuronoxylan, in this EDTA-soluble extract. These radioactively labeled polysaccha-rides were found to bind to cell wall ghosts and to xyloglucan extracted from the second and third internodes of pea epicotyls cell walls in a pH-dependent manner, similar to the binding pattern obtained with nascent polymers. Maxi-mum binding occurred at pH 3-4, and also required the presence of protein.
