Translation of messenger RNAs lacking a stop codon results in the addition of a carboxy-terminal poly-lysine tract to the nascent polypeptide, causing ribosome stalling. Non-stop proteins and other stalled nascent chains are recognized by the ribosome quality control (RQC) machinery and targeted for proteasomal degradation. Failure of this process leads to neurodegeneration by unknown mechanisms. Here we show that deletion of the E3 ubiquitin ligase Ltn1p in yeast, a key RQC component, causes stalled proteins to form detergent-resistant aggregates and inclusions. Aggregation is dependent on a C-terminal alanine/threonine tail that is added to stalled polypeptides by the RQC component, Rqc2p. Formation of inclusions additionally requires the poly-lysine tract present in non-stop proteins. The aggregates sequester multiple cytosolic chaperones and thereby interfere with general protein quality control pathways. These findings can explain the proteotoxicity of ribosome-stalled polypeptides and demonstrate the essential role of the RQC in maintaining proteostasis.

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The bacterial phosphoenolpyruvate:sugar phosphotransferase system regulates a variety of physiological processes as well as effecting sugar transport. The crr gene product (enzyme IIA Glc (IIA Glc )) mediates some of these regulatory phenomena. In this report, we characterize a novel IIA Glc -binding protein from Escherichia coli extracts, discovered using ligand-fishing with surface plasmon resonance spectroscopy. This protein, which we named FrsA (fermentation/respiration switch protein), is the 47-kDa product of the yafA gene, previously denoted as "function unknown." FrsA forms a 1:1 complex specifically with the unphosphorylated form of IIA Glc , with the highest affinity of any protein thus far shown to interact with IIA Glc . Orthologs of FrsA have been found to exist only in facultative anaerobes belonging to the ␥-proteobacterial group. Disruption of frsA increased cellular respiration on several sugars including glucose, while increased FrsA expression resulted in an increased fermentation rate on these sugars with the concomitant accumulation of mixed-acid fermentation products. These results suggest that IIA Glc regulates the flux between respiration and fermentation pathways by sensing the available sugar species via a phosphorylation state-dependent interaction with FrsA. The bacterial phosphoenolpyruvate(PEP)1 :sugar phosphotransferase system (PTS) plays an important role in the transport of a variety of sugar substrates. This system catalyzes phosphorylation coupled to translocation of numerous simple sugars across the cytoplasmic membrane. The PTS is composed of two general cytoplasmic proteins, enzyme I (EI) and histidine phosphocarrier protein, HPr, which are used for all sugars, and, in addition, some sugar-specific components collectively known as enzymes II (1). The glucose-specific enzyme II of Escherichia coli consists of two components: soluble enzyme IIA Glc (IIA Glc ) and membrane-bound enzyme IICB Glc (IICB Glc ). Thus, glucose transport in E. coli involves three soluble PTS components (EI, HPr, and IIA Glc , encoded by the ptsHIcrr operon) and one membrane-bound protein, enzyme IICB Glc (encoded by the ptsG gene). Glucose uptake entails sequential phosphoryl transfer via the PTS, as follows: phosphoenolpyruvate (PEP) 3 EI 3 HPr 3 IIA Glc 3 IICB Glc 3 glucose. Components of the PTS also participate in several regulatory mechanisms (1). Catabolite repression allows for the preferential utilization of sugars transported by the PTS. Consequently, when E. coli are cultured in a medium containing both glucose and a non-PTS sugar, the glucose is consumed first. The currently accepted mechanism for this effect is that, when PTS sugars are transported, the steady-state condition of IIA Glc is mainly in the dephospho-form. The unphosphorylated form of IIA Glc inhibits transport of non-PTS sugars such as lactose, maltose, melibiose, and raffinose by interacting with transporters for these sugars (a process termed inducer exclusion) (1, 2-4). Other allosteric regulatory functions of IIA Glc incl...

alpha-Synuclein is a pathological component of PD (Parkinson's disease) by participating in Lewy body formation. JC-1 (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide) has been shown to interact with alpha-synuclein at the acidic C-terminal region with a K(d) of 2.6 microM. JC-1 can discriminated between the fibrillation states of alpha-synuclein (monomeric, oligomeric intermediate and fibrillar forms) by emitting the enhanced binding fluorescence of different colours at 590, 560 and 538 nm respectively with the common excitation at 490 nm. The fibrillation-state-specific interaction of JC-1 allowed us to perform real-time analyses of the alpha-synuclein fibrillation in the presence of iron as a fibrillation inducer, rifampicin as a fibrillation inhibitor, baicalein as a defibrillation agent and dequalinium as a protofibril inducer. In addition, various alpha-synuclein fibrils with different morphologies prepared with specific ligands such as metal ions, glutathione, eosin and lipids were monitored with their characteristic JC-1-binding fluorescence spectra. FRET (fluorescence resonance energy transfer) between thioflavin-T and JC-1 was also employed to specifically identify the amyloid fibrils of alpha-synuclein. Taken together, we have introduced JC-1 as a powerful and versatile probe to explore the molecular mechanism of the fibrillation process of alpha-synuclein in vitro. It could be also useful in high-throughput drug screening. The specific alpha-synuclein interaction of JC-1 would therefore contribute to our complete understanding of the molecular aetiology of PD and eventual development of diagnostic/therapeutic strategies for various alpha-synucleinopathies.