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. In the case of native RTA, we established that dislocation also depends on other components of the classical ERAD-L pathway as well as an ongoing ER-Golgi transport. However, the dislocation pathways deviate strikingly upon entry into the cytosol. Here, the CDC48 complex is required only for RTA(Delta), although the involvement of individual ATPases (Rpt proteins) in the 19S regulatory particle (RP) of the proteasome, and the 20S catalytic chamber itself, is very different for the two RTA variants. We conclude that cytosolic ERAD components, particularly the proteasome RP, can discriminate between structural features of the same substrate.

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. A subsequent switch in conditions to provoke innate toxin action would permit only those strains containing defects in genes normally essential for toxin retro-translocation, refolding or degradation to survive. As a route to such a screen, several RTA mutants with reduced catalytic activity have previously been isolated. Here we report the use of Saccharomyces cerevisiae to isolate temperature-dependent mutants of endoplasmic reticulum-targeted RTA. Two such toxin mutants with opposing phenotypes were isolated. One mutant RTA (RTAF108L/L151P) allowed the yeast cells that express it to grow at 37 degrees C, whereas the same cells did not grow at 23 degrees C. Both mutations were required for temperature-dependent growth. The second toxin mutant (RTAE177D) allowed cells to grow at 23 degrees C but not at 37 degrees C. Interestingly, RTAE177D has been previously reported to have reduced catalytic activity, but this is the first demonstration of a temperature-sensitive phenotype. To provide a more detailed characterization of these mutants we have investigated their N-glycosylation, stability, catalytic activity and, where appropriate, a three-dimensional structure. The potential utility of these mutants is discussed.

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. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates.

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). Mim-1 has similarities to the conserved acetyltransferase motifs found in the GNAT superfamily of proteins and also contains three minimal GK acetylation motifs. These data identify Mim-1 as an acetyltransferase.

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. In this study we have tested the importance of conserved residues on the periplasmic and cytoplasmic face of the Escherichia coli protein. We find that many of the mutations on the cytoplasmic face have little or no effect. However, substitution at several positions in the extreme N-terminal cytoplasmic region or the predicted first cytoplasmic loop lead to a significant or complete loss of Tat-dependent export. The mutated strains are unable to grow anaerobically on trimethylamine N-oxide minimal media and are unable to export trimethylamine-N-oxide reductase (TorA). The same mutants are completely unable to export a chimeric protein, comprising the TorA signal peptide linked to green fluorescent protein, indicating that translocation is blocked rather than cofactor insertion into the TorA mature protein. The data point to two essential cytoplasmic domains on the TatC protein that are essential for export.