Summary: Small heat shock proteins (sHsps) can efficiently prevent the aggregation of unfolded proteins in vitro. However, how this in vitro activity translates to function in vivo is poorly understood. We demonstrate that sHsps of Escherichia coli, IbpA and IbpB, co‐operate with ClpB and the DnaK system in vitro and in vivo, forming a functional triade of chaperones. IbpA/IbpB and ClpB support independently and co‐operatively the DnaK system in reversing protein aggregation. A ΔibpABΔclpB double mutant exhibits strongly increased protein aggregation at 42°C compared with the single mutants. sHsp and ClpB function become essential for cell viability at 37°C if DnaK levels are reduced. The DnaK requirement for growth is increasingly higher for ΔibpAB, ΔclpB, and the double ΔibpABΔclpB mutant cells, establishing the positions of sHsps and ClpB in this chaperone triade.

Disruption of the functional protein balance in living cells activates protective quality control systems to repair damaged proteins or sequester potentially cytotoxic misfolded proteins into aggregates. The established model based on Saccharomyces cerevisiae indicates that aggregating proteins in the cytosol of eukaryotic cells partition between cytosolic juxtanuclear (JUNQ) and peripheral deposits. Substrate ubiquitination acts as the sorting principle determining JUNQ deposition and subsequent degradation. Here, we show that JUNQ unexpectedly resides inside the nucleus, defining a new intranuclear quality control compartment, INQ, for the deposition of both nuclear and cytosolic misfolded proteins, irrespective of ubiquitination. Deposition of misfolded cytosolic proteins at INQ involves chaperone‐assisted nuclear import via nuclear pores. The compartment‐specific aggregases, Btn2 (nuclear) and Hsp42 (cytosolic), direct protein deposition to nuclear INQ and cytosolic (CytoQ) sites, respectively. Intriguingly, Btn2 is transiently induced by both protein folding stress and DNA replication stress, with DNA surveillance proteins accumulating at INQ. Our data therefore reveal a bipartite, inter‐compartmental protein quality control system linked to DNA surveillance via INQ and Btn2. Synopsis ‘Juxtanuclear’ deposits of misfolded protein in fact reside in a new intranuclear quality control compartment (INQ). INQ formation is dependent on Btn2, while cytosolic protein deposition is mediated by the Hsp42 aggregase. Proteotoxic stress in yeast results in organized sequestration of misfolded proteins at cytosolic CytoQ and nuclear INQ (former JUNQ) deposition sites. INQ deposition does not rely on substrate ubiquitination and does not target sequestered proteins for degradation. CytoQ and INQ formation relies on compartment‐specific aggregases, the cytosolic Hsp42 and the nuclear Btn2. DNA replication stress triggers Btn2‐dependent protein sequestration, linking cellular mechanisms coping with protein and DNA stress.

The recently identified type VI secretion systems (T6SS) have a crucial function in the virulence of various proteobacteria, including the human pathogen Vibrio cholerae . T6SS are encoded by a conserved gene cluster comprising approximately 15 open reading frames, mediating the appearance of Hcp and VgrG proteins in cell culture supernatants. Here, we analysed the function of the V. cholerae T6SS member ClpV, a specialized AAA+ protein. ClpV is crucial for a functional T6SS and interacts through its N‐terminal domain with the VipA/VipB complex that is composed of two conserved and essential members of T6SS. Transferring ClpV substrate specificity to a distinct AAA+ protein involved in proteolysis caused degradation of VipA but not Hcp or VgrG2, suggesting that VipA rather than Hcp/VgrG2 functions as a primary ClpV substrate. Strikingly, VipA/VipB form tubular, cogwheel‐like structures that are converted by a threading activity of ClpV into small complexes. ClpV‐mediated remodelling of VipA/VipB tubules represents a crucial step in T6S, illuminating an unexpected role of an ATPase component in protein secretion. There is a [Have you seen ...?][1] (February 2009) associated with this Article. [1]: http://dx.doi.org/10.1038/emboj.2008.301

The aggregation of proteins inside cells is an organized process with cytoprotective function. In Saccharomyces cerevisiae, aggregating proteins are spatially sequestered to either juxtanuclear or peripheral sites, which target distinct quality control pathways for refolding and degradation. The cellular machinery driving the sequestration of misfolded proteins to these sites is unknown. In this paper, we show that one of the two small heat shock proteins of yeast, Hsp42, is essential for the formation of peripheral aggregates during physiological heat stress. Hsp42 preferentially localizes to peripheral aggregates but is largely absent from juxtanuclear aggregates, which still form in hsp42Δ cells. Transferring the amino-terminal domain of Hsp42 to Hsp26, which does not participate in aggregate sorting, enables Hsp26 to replace Hsp42 function. Our data suggest that Hsp42 acts via its amino-terminal domain to coaggregate with misfolded proteins and perhaps link such complexes to further sorting factors.