RPS9

In Toxoplasma gondii, RPS9 is a nuclear-encoded apicoplast ribosomal protein that carries a bipartite N-terminal leader directing it into the apicoplast through a multi-step processing pathway (PMID:11704269). The first 55 amino acids of this leader are sufficient for apicoplast targeting, and processing of the transit peptide occurs as a multi-step event both outside of and at/within the organelle (PMID:11704269). Two discrete cis-acting regions within the transit domain (amino acids 38–49 and 79–86), acting together with the signal sequence, are necessary and sufficient for targeting (PMID:14533689). During transit, the leader physically associates with subtilisin-like proteases (92- and 90-kDa species) and the T. gondii molecular chaperone BiP (86-kDa species), implicating these factors in leader processing and/or import (PMID:14533689). Beyond this targeting and processing pathway, the biochemical function of RPS9 within the assembled apicoplast ribosome has not been characterized in the available corpus.

1. 2001 Medium

   Whether the RPS9 leader alone could route a nuclear-encoded protein to the apicoplast, and whether transit peptide removal was a single or multi-step event, was unknown; defining the minimal targeting determinant established the logic of apicoplast protein import.

   Evidence Deletional/insertional mutagenesis with HA tagging, fluorescence localization, and Western blotting in transfected T. gondii

   PMID:11704269

   Open questions at the time

   • The specific proteases or chaperones mediating each processing step were not identified
   • The precise cleavage sites within the leader were not mapped
   • Function of mature RPS9 in the apicoplast ribosome was not addressed
2. 2003 Medium

   Having localized targeting to the first 55 residues, the next question was which specific sequence elements act as cis determinants and which trans factors engage the leader; mapping two transit-domain regions and identifying associated proteins built the molecular basis of leader recognition and processing.

   Evidence Cis-element mutagenesis, GST-fusion affinity chromatography, and mass spectrometry of leader-associated proteins in T. gondii transfectants

   PMID:14533689

   Open questions at the time

   • Direct enzymatic activity of the subtilisin-like proteases on the RPS9 leader was not demonstrated
   • The functional role of BiP association during import was not tested by loss-of-function
   • The 160-kDa associated protein was not identified

• The function of mature RPS9 within the apicoplast ribosome and the mechanistic role of the associated proteases and BiP in import remain unresolved.
• No structural or functional characterization of RPS9 in the assembled ribosome
• No loss-of-function or reconstitution of the protease/chaperone-mediated import step