Folding-competent and folding-defective forms of ricin A chain have different fates after retrotranslocation from the endoplasmic reticulum.

We report that a toxic polypeptide retaining the potential to refold upon dislocation from the endoplasmic reticulum (ER) to the cytosol (ricin A chain; RTA) and a misfolded version that cannot (termed RTA(Delta)), follow ER-associated degradation (ERAD) pathways in Saccharomyces cerevisiae that substantially diverge in the cytosol. Both polypeptides are dislocated in a step mediated by the transmembrane Hrd1p ubiquitin ligase complex and subsequently degraded. Canonical polyubiquitylation is not a prerequisite for this interaction because a catalytically inactive Hrd1p E3 ubiquitin ligase retains the ability to retrotranslocate RTA, and variants lacking one or both endogenous lysyl residues also require the Hrd1p complex.

The isolation and characterization of temperature-dependent ricin A chain molecules in Saccharomyces cerevisiae.

Ricin is a heterodimeric plant protein that is potently toxic to mammalian cells. Toxicity results from the catalytic depurination of eukaryotic ribosomes by ricin toxin A chain (RTA) that follows toxin endocytosis to, and translocation across, the endoplasmic reticulum membrane. To ultimately identify proteins required for these later steps in the entry process, it will be useful to express the catalytic subunit within the endoplasmic reticulum of yeast cells in a manner that initially permits cell growth.

Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.

Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants.

Myb induced myeloid protein 1 (Mim-1) is an acetyltransferase.

We have screened protein extracts from chicken blood cells for acetyltransferases. An in gel acetyltransferase assay revealed that a 32 kDa protein, which is more prevalent in whole blood when compared with erythrocyte cells, possessed an auto-acetylation activity. This protein was purified by a series of chromatographic steps, sequenced by Edman degradation and subsequently identified as Myb induced myeloid protein (Mim-1).

Essential cytoplasmic domains in the Escherichia coli TatC protein.

The twin-arginine translocation (Tat) system mediates the transport of proteins across the bacterial plasma membrane and chloroplast thylakoid membrane. Operating in parallel with Sec-type systems in these membranes, the Tat system is completely different in both structural and mechanistic terms, and is uniquely able to catalyze the translocation of fully folded proteins across coupled membranes. TatC is an essential, multispanning component that has been proposed to form part of the binding site for substrate precursor proteins.