Intracellular trafficking of ricin A chain in relation to its cytotoxicity and depurination in Saccharomyces cerevisiae
Creators: name: Yan, Qing
Publisher: name: No Publisher Supplied
Year: 2013

description: Ricin is a heterodimeric protein composed of ricin A chain (RTA) and ricin B chain (RTB). The extreme toxicity of ricin is mainly attributed to the enzymatically active RTA, which specifically removes an adenine residue from the universally conserved α-sarcin/ricin loop (SRL) in the 28S rRNA and inhibits protein synthesis. For RTA to exert its depurination activity, it has to undergo retrograde transport pathway to enter the cytosol. Therefore, the intracellular trafficking of RTA is an important aspect of ricin mediated cell death. The goal of the present study is to understand the structural features of RTA which contribute to its intracellular transport and the host factors involved in its trafficking in Saccharomyces cerevisiae. To visualize RTA, the enhanced green fluorescent protein (EGFP) is fused to mature RTA (matRTA-EGFP) containing 26&amp;-amino acid residues and precursor RTA (preRTA-EGFP) containing a 35-amino acid N-terminal extension followed by mature RTA. When preRTA-EGFP is expressed in the endoplasmic reticulum (ER) lumen of Saccharomyces cerevisiae, it follows two parallel pathways: 1) the well characterized ER-to-cytosol dislocation through the ER-associated degradation (ERAD) pathway, 2) vacuole transport after initial localization to the ER. In chapter two, I studied the roles of the important structural domains and sequence motifs of RTA in its trafficking and toxicity by mutational analysis. I showed that the 26-amino acid signal peptide within the N-terminal extension of preRTA is responsible for the ER targeting and the following nine-amino acid propeptide is important for glycosylation and efficient vacuole transport. The N-glycosylation of RTA promotes efficient ER export to the cytosol and vacuole, and contributes to the toxicity of RTA. The C-terminal hydrophobic domain of RTA is critical for transport out of the ER. In chapter three, I further investigated how the ER-to-vacuole transport and ER-to-cytosol dislocation pathways of preRTA affect its depurination and toxicity by using wild type preRTA and nontoxic preRTA mutants. The altered sequence motifs in preRTA lead to different translocation pathways. This provides the advantage of studying the contribution of their trafficking pathways to the toxicity of RTA. I present evidence that vacuole transport is an alternative degradation pathway of preRTA and contributes to the depurination reduction. The dislocated wild type preRTA and preRTA mutants have differential requirements for the cytosolic ERAD component peptide:N-glycanase (PNGase; yeast Png1), which is involved in the degradation of its substrates. In chapter four, the roles of yeast cellular components in the trafficking of RTA were studied to unravel the host genes in the ricin trafficking pathways. A genome wide screen of yeast nonessential gene knockout collection against RTA led to the discovery of genes in the Hrd1p complex and AP-2 complex, whose deletions conferred resistance to preRTA but not to matRTA. The components of the Hrd1p complex are responsible for the dislocation of preRTA. The Apl3p subunit of AP-2 complex is involved in the vacuole transport of preRTA. The study has provided important insights into the mechanism of intracellular trafficking of RTA and its role in toxicity.