Balanced activities of Hsp70 and the ubiquitin proteasome system underlie cellular protein homeostasis.

To counteract proteotoxic stress and cellular aging, protein quality control (PQC) systems rely on the refolding, degradation and sequestration of misfolded proteins. In Saccharomyces cerevisiae the Hsp70 chaperone system plays a central role in protein refolding, while degradation is predominantly executed by the ubiquitin proteasome system (UPS). The sequestrases Hsp42 and Btn2 deposit misfolded proteins in cytosolic and nuclear inclusions, thereby restricting the accessibility of misfolded proteins to Hsp70 and preventing the exhaustion of limited Hsp70 resources. Therefore, in yeast, sequestrase mutants show negative genetic interactions with double mutants lacking the Hsp70 co-chaperone Fes1 and the Hsp104 disaggregase (fes1Δ hsp104Δ, ΔΔ) and suffering from low Hsp70 capacity. Growth of ΔΔbtn2Δ mutants is highly temperature-sensitive and results in proteostasis breakdown at non-permissive temperatures. Here, we probed for the role of the ubiquitin proteasome system in maintaining protein homeostasis in ΔΔbtn2Δ cells, which are affected in two major protein quality control branches. We show that ΔΔbtn2Δ cells induce expression of diverse stress-related pathways including the ubiquitin proteasome system to counteract the proteostasis defects. Ubiquitin proteasome system dependent degradation of the stringent Hsp70 substrate firefly Luciferase in the mutant cells mirrors such compensatory activities of the protein quality control system. Surprisingly however, the enhanced ubiquitin proteasome system activity does not improve but aggravates the growth defects of ΔΔbtn2Δ cells. Reducing ubiquitin proteasome system activity in the mutant by lowering the levels of functional 26S proteasomes improved growth, increased refolding yield of the Luciferase reporter and attenuated global stress responses. Our findings indicate that an imbalance between Hsp70-dependent refolding, sequestration and ubiquitin proteasome system-mediated degradation activities strongly affects protein homeostasis of Hsp70 capacity mutants and contributes to their severe growth phenotypes.

Cellular sequestrases maintain basal Hsp70 capacity ensuring balanced proteostasis.

Maintenance of cellular proteostasis is achieved by a multi-layered quality control network, which counteracts the accumulation of misfolded proteins by refolding and degradation pathways. The organized sequestration of misfolded proteins, actively promoted by cellular sequestrases, represents a third strategy of quality control. Here we determine the role of sequestration within the proteostasis network in Saccharomyces cerevisiae and the mechanism by which it occurs. The Hsp42 and Btn2 sequestrases are functionally intertwined with the refolding activity of the Hsp70 system. Sequestration of misfolded proteins by Hsp42 and Btn2 prevents proteostasis collapse and viability loss in cells with limited Hsp70 capacity, likely by shielding Hsp70 from misfolded protein overload. Btn2 has chaperone and sequestrase activity and shares features with small heat shock proteins. During stress recovery Btn2 recruits the Hsp70-Hsp104 disaggregase by directly interacting with the Hsp70 co-chaperone Sis1, thereby shunting sequestered proteins to the refolding pathway.

A prion-like domain in Hsp42 drives chaperone-facilitated aggregation of misfolded proteins.

Chaperones with aggregase activity promote and organize the aggregation of misfolded proteins and their deposition at specific intracellular sites. This activity represents a novel cytoprotective strategy of protein quality control systems; however, little is known about its mechanism. In yeast, the small heat shock protein Hsp42 orchestrates the stress-induced sequestration of misfolded proteins into cytosolic aggregates (CytoQ). In this study, we show that Hsp42 harbors a prion-like domain (PrLD) and a canonical intrinsically disordered domain (IDD) that act coordinately to promote and control protein aggregation. Hsp42 PrLD is essential for CytoQ formation and is bifunctional, mediating self-association as well as binding to misfolded proteins. Hsp42 IDD confines chaperone and aggregase activity and affects CytoQ numbers and stability in vivo. Hsp42 PrLD and IDD are both crucial for cellular fitness during heat stress, demonstrating the need for sequestering misfolded proteins in a regulated manner.

Small heat shock proteins sequester misfolding proteins in near-native conformation for cellular protection and efficient refolding.

Small heat shock proteins (sHsp) constitute an evolutionary conserved yet diverse family of chaperones acting as first line of defence against proteotoxic stress. sHsps coaggregate with misfolded proteins but the molecular basis and functional implications of these interactions, as well as potential sHsp specific differences, are poorly explored. In a comparative analysis of the two yeast sHsps, Hsp26 and Hsp42, we show in vitro that model substrates retain near-native state and are kept physically separated when complexed with either sHsp, while being completely unfolded when aggregated without sHsps. Hsp42 acts as aggregase to promote protein aggregation and specifically ensures cellular fitness during heat stress. Hsp26 in contrast lacks aggregase function but is superior in facilitating Hsp70/Hsp100-dependent post-stress refolding. Our findings indicate the sHsps of a cell functionally diversify in stress defence, but share the working principle to promote sequestration of misfolding proteins for storage in native-like conformation.

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

Compartment-specific aggregases direct distinct nuclear and cytoplasmic aggregate deposition.

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.

Fabrication of optical filters based on polymer asymmetric Bragg couplers.

In this work, we successfully developed a process to fabricate dual-channel polymeric waveguide filters based on an asymmetric Bragg coupler (ABC) using holographic interference techniques, soft lithography, and micro molding. At the cross- and self-reflection Bragg wavelengths, the transmission dips of approximately -16.4 and -11.5 dB relative to the 3 dB background insertion loss and the 3 dB transmission bandwidths of approximately 0.6 and 0.5 nm were obtained from an ABC-based filter. The transmission spectrum overlaps when the effective index difference between two single waveguides is less than 0.002.

Fabrication of high-resolution periodical structure on polymer waveguides using a replication process.

This paper describes a procedure to replicate a polymeric wavelength filter. In this work, the grating structure on a polymer is fabricated first using holographic interferometry and micro-molding processes. The polymeric wavelength filters are produced by a two-step molding process where the master mold is first formed on a negative tone photoresist and subsequently transferred to a PDMS mold; following this step, the PDMS silicon rubber mold was used as a stamp to transfer the pattern of the polymeric wavelength filters onto a UV cure epoxy. Initial results show good pattern transfer in physical shape. At the Bragg wavelength, a transmission dip of -15.5 dB relative to the -3dB background insertion loss and a 3-dB-transmission bandwidth of ?6nm were obtained from the device.

Fabrication of polymer waveguides by a replication method.

We have developed a soft-lithography method to replicate polymer waveguides. In this method, the waveguides are produced by a two-step molding process where a master mold is first formed on a negative-tone photoresist and subsequently transferred to a polydimethylsiloxane (PDMS) mold; a PDMS silicone rubber mold is then used as a stamp to transfer the final waveguide pattern onto an UV cure epoxy. Initial results show good pattern transferring in physical shape. The optical performance is measured based on the propagation loss. In our design, the loss was measured at 0.28 dB/cm for 1.3 microm and 0.26 dB/cm for 1.55 microm.

Transducing mechanical force by use of a diffraction grating sensor.

A novel means of transducing mechanical force by using a polymeric-based diffractive grating sensor is presented. The diffraction gratings are successfully fabricated upon poly(dimethyl siloxane) polymer substrates by holographic interference and micromolding. A micromaterial tensile test incorporated into the surface diffraction grating experiment showed that a relationship between the load and the observed diffraction-pattern shift could be obtained. The results show an excellent correlation between the optical measurement and load, with a sensitivity of 0.05 N.

Fabrication of a high-resolution periodical structure using a replication process.

We describe a procedure for rapidly and conveniently prototyping a periodic structure at submicrometer order using holographic interferometry and micro-molding processes. In this experiment, the master of the periodic structure was created on an i-line submicrometer positive photoresist film by a holographic interference using a He-Cd (325nm) laser. A subsequent mold using polydimethylsiloxane (PDMS) polymer was cast against this master and used as a stamp to transfer the grating pattern onto a UV cure epoxy. The technique shows accurate control for the transferring of a grating's period and depth. The grating pattern on the epoxy produced by the PDMS mold shows an average of less than 2% error in the grating period and an average of 15% error in depth reproduction.