An In Vitro Assay for Substrate Translocation by FhaC in Liposomes.

The two-partner secretion (TPS) pathway is used by gram-negative bacteria to secrete a large family of virulence exoproteins. Its name is derived from the fact that it involves two proteins, a secreted TpsA protein and a cognate TpsB transporter in the outer membrane. A typical TPS system is represented by the filamentous hemagglutinin FhaB (TpsA protein) and its transporter FhaC (TpsB protein) of Bordetella pertussis. Results from mutational analysis and heterologous expression experiments suggested that FhaC is essential for FhaB translocation across the outer membrane of bacteria. We have devised a cell-free biochemical assay to reconstitute in vitro the translocation of FhaB into reconstituted membrane vesicles. Thereby the clearest evidence has been provided that the single ß-barrel FhaC protein serves as the sole translocator to transport FhaB across the outer membrane. This is the first in vitro assay for protein secretion across the Escherichia coli outer membrane and the detailed protocol described here should be amenable to modifications and application to the analysis of related protein transport events occurring at the outer membranes of gram-negative bacteria.

Absence of the Yeast Hsp31 Chaperones of the DJ-1 Superfamily Perturbs Cytoplasmic Protein Quality Control in Late Growth Phase.

The Saccharomyces cerevisiae heat shock proteins Hsp31, Hsp32, Hsp33 and Hsp34 belong to the DJ-1/ThiJ/PfpI superfamily which includes the human protein DJ-1 (PARK7) as the most prominent member. Mutations in the DJ-1 gene are directly linked to autosomal recessive, early-onset Parkinson's disease. DJ-1 acts as an oxidative stress-induced chaperone preventing aggregation and fibrillation of α-synuclein, a critical factor in the development of the disease. In vivo assays in Saccharomyces cerevisiae using the model substrate ΔssCPY*Leu2myc (ΔssCL*myc) as an aggregation-prone misfolded cytoplasmic protein revealed an influence of the Hsp31 chaperone family on the steady state level of this substrate. In contrast to the ubiquitin ligase of the N-end rule pathway Ubr1, which is known to be prominently involved in the degradation process of misfolded cytoplasmic proteins, the absence of the Hsp31 chaperone family does not impair the degradation of newly synthesized misfolded substrate. Also degradation of substrates with strong affinity to Ubr1 like those containing the type 1 N-degron arginine is not affected by the absence of the Hsp31 chaperone family. Epistasis analysis indicates that one function of the Hsp31 chaperone family resides in a pathway overlapping with the Ubr1-dependent degradation of misfolded cytoplasmic proteins. This pathway gains relevance in late growth phase under conditions of nutrient limitation. Additionally, the Hsp31 chaperones seem to be important for maintaining the cellular Ssa Hsp70 activity which is important for Ubr1-dependent degradation.

Versatile in vitro system to study translocation and functional integration of bacterial outer membrane proteins.

Gram-negative bacteria use the type-V secretion pathway to expose proteins at their cell surface, many of which have virulence functions. Translocation of those proteins across the outer membrane occurs either by means of dedicated translocator proteins (two-partner secretion) or covalently fused translocator domains (autotransporters). Translocator proteins and translocator domains are ß-barrels requiring the ß-barrel assembly machinery (BAM) for membrane integration. However, the molecular details of their passage across the envelope and insertion into the outer membrane remain enigmatic, owing in part to the fact that in vitro systems are not available. Here we describe a versatile in vitro reconstitution system that faithfully reproduces both branches of the type-V secretion pathway and the assembly of ß-barrel outer membrane proteins. This system will allow an in-depth analysis of protein secretion across and integration into outer membranes.