Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting.
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 15 degrees C, 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.
DeRocher
Amy
A
Seattle Biomedical Research Institute, 307 Westlake Avenue N., Suite 500 Seattle, WA 98109, USA.
Gilbert
Brian
B
Feagin
Jean E
JE
Parsons
Marilyn
M
D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology*; D003080:Cold Temperature; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D009842:Oligopeptides / Q000235:genetics; D018087:Plastids / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014122:Toxoplasma / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
D000213:Acyl Carrier Protein; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000091243:Ribosomal Protein S9; D012269:Ribosomal Proteins; C077480:histidyl-aspartyl-glutamyl-leucine; D049452:Green Fluorescent Proteins; D020126:Brefeldin A