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 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.
Yung
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S
Division of Geographic Medicine, BBRB 203, University of Alabama at Birmingham, 1530 3rd Avenue South, Birmingham, AL 35294-2170, USA.
Unnasch
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TR
Lang-Unnasch
N
N
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D018274:Electroporation; D017353:Gene Deletion; D006801:Humans; D008856:Microscopy, Fluorescence; D016254:Mutagenesis, Insertional; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D018087:Plastids / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals; D021122:Protein Subunits; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism*; D014122:Toxoplasma / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014162:Transfection
D010455:Peptides; D021382:Protein Sorting Signals; D021122:Protein Subunits; D015800:Protozoan Proteins; D012269:Ribosomal Proteins