Analysis of apicoplast targeting and transit peptide processing in Toxoplasma gondii by deletional and insertional mutagenesis

Deletion and insertion mutagenesis was used to analyze the targeting sequence of the nuclear encoded apicoplast protein, the ribosomal protein small subunit 9 of Toxoplasma gondii. Previous studies have shown that nuclear encoded apicoplast proteins possess bipartite leaders having characteristic si...

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Bibliographic Details
Published in:Molecular and biochemical parasitology 2001-11, Vol.118 (1), p.11-21
Main Authors: Yung, Sunny, Unnasch, Thomas R, Lang-Unnasch, Naomi
Format: Article
Language:English
Subjects:
Animals
Apicoplast
apicoplasts
Blotting, Western
Cell Line
Electroporation
Gene Deletion
Humans
Microscopy, Fluorescence
Mutagenesis, Insertional
Peptides - genetics
Peptides - metabolism
Plasmids - genetics
Plastids - genetics
Plastids - metabolism
Protein Sorting Signals
Protein Subunits
Protein targeting
Protein Transport
Protozoan Proteins - genetics
Protozoan Proteins - metabolism
Ribosomal protein
Ribosomal Proteins - genetics
Ribosomal Proteins - metabolism
rRNA 9S
Toxoplasma - genetics
Toxoplasma - growth & development
Toxoplasma - metabolism
Toxoplasma gondii
Transfection
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Summary:Deletion and insertion mutagenesis was used to analyze the targeting sequence of the nuclear encoded apicoplast protein, the ribosomal protein small subunit 9 of Toxoplasma gondii. Previous studies have shown that nuclear encoded apicoplast proteins possess bipartite leaders having characteristic signal sequences followed by serine/threonine rich transit sequences. Deletion analysis demonstrated that the first 55 amino acids of the rps9 leader were sufficient for apicoplast targeting. Insertional mutagenesis tagging the leader sequence with a hemagglutinin (HA) tag was used to study the events involved in the targeting pathway. Transfectants with insertions near the N-terminus of the transit displayed HA tagged precursors outside of the apicoplast, in the perinuclear region. In contrast, transfectants with the HA tag inserted near the carboxyl end of the transit-like region had apicoplast labeling. Western blot analysis of HA tagged stable isolates suggested that processing of the HA tagged leaders was a multi-step process, with processing occurring both outside of and at or within the apicoplast.
ISSN:0166-6851
1872-9428
DOI:10.1016/S0166-6851(01)00359-0