Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

被引:51
作者
DeRocher, A
Gilbert, B
Feagin, JE
Parsons, M
机构
[1] Seattle Biomed Res Inst, Seattle, WA 98109 USA
[2] Univ Washington, Sch Publ Hlth & Community Med, Dept Pathobiol, Seattle, WA 98195 USA
关键词
toxoplasma; apicoplast; chloroplast; brefeldin A; endoplasmic reticulum; Apicomplexa;
D O I
10.1242/jcs.01627
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
The apicoplast is a relict plastid found in many apicomplexans, including the pathogens Toxoplasma gondii and Plasmodium falciparum. Nucleus-encoded apicoplast proteins enter the ER, and after cleavage of the signal sequence, are routed to the apicoplast by virtue of a transit peptide, which is subsequently removed. To assess the mechanisms of localization we examined stable transfectants of T. gondii for the localization and processing of various GFP fusion proteins. GFP fusions bearing apicoplast targeting sequences targeted efficiently to the plastid, with no retention in the ER, even when an ER retention/retrieval sequence was added. Incubation with brefeldin A, which blocks ER-to-Golgi trafficking by inhibiting a GTP exchange factor required for retrograde trafficking, blocked the processing of the protein. Surprisingly, it did not affect the immunofluorescence pattern. To avoid the potentially misleading presence of pre-existing GFP fusion protein in the apicoplast, we used a ligand-regulated aggregation system to arrest the GFP fusion protein in the ER prior to trafficking. Upon addition of ligand to promote disaggregation, the fusion protein targeted to the plastid, even in the presence of brefeldin A. Ligand release at 15degreesC, which blocks trafficking of Golgi-routed proteins, also allowed significant localization to the plastid. Our data indicate that apicoplast proteins can localize to the region of the plastid when Golgi trafficking is inhibited, but suggest that some steps in import or maturation of the proteins may require a brefeldin A-sensitive GTP exchange factor.
引用
收藏
页码:565 / 574
页数:10
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