title: N-glycosylation does not affect the catalytic activity of ricin a chain but stimulates cytotoxicity by promoting its transport out of the endoplasmic reticulum.
abstract: Ricin A chain (RTA) depurinates the α-sarcin/ricin loop after it undergoes retrograde trafficking to the cytosol. The structural features of RTA involved in intracellular transport are not known. To explore this, we fused enhanced green fluorescent protein (EGFP) to precursor (preRTA-EGFP), containing a 35-residue leader, and mature RTA (matRTA-EGFP). Both were enzymatically active and toxic in Saccharomyces cerevisiae. PreRTA-EGFP was localized in the endoplasmic reticulum (ER) initially and was subsequently transported to the vacuole, whereas matRTA-EGFP remained in the cytosol, indicating that ER localization is a prerequisite for vacuole transport. When the two glycosylation sites in RTA were mutated, the mature form was fully active and toxic, suggesting that the mutations do not affect catalytic activity. However, nonglycosylated preRTA-EGFP had reduced toxicity, depurination and delayed vacuole transport, indicating that N-glycosylation affects transport of RTA out of the ER. Point mutations in the C-terminal hydrophobic region restricted RTA to the ER and eliminated toxicity and depurination, indicating that this sequence is critical for ER exit. These results demonstrate that N-glycosylation and the C-terminal hydrophobic region stimulate the toxicity of RTA by promoting ER export. The timing of depurination coincided with the timing of vacuole transport, suggesting that RTA may enter the cytosol during vacuole transport.
authors.affiliation: Department of Plant Biology and Pathology, School of Environmental and Biological Sciences, Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901-8520, USA.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yan
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Li
authors.forename: Nilgun E
authors.initials: NE
authors.lastname: Tumer
mesh_terms: D020816:Amino Acid Motifs; D020134:Catalytic Domain; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics; D017354:Point Mutation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012276:Ricin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
chemical_list: D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012276:Ricin