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The Small Heat Shock Protein Hsp42 Controls the Spatio-Temporal Organization of Aggregated Proteins in Saccharomyces Cerevisiae
Stress-induced protein aggregation represents a major threat for cell survival and is also associated with various human disorders and cellular aging. The primary cellular response to aberrant protein conformations is the refolding of misfolded proteins by molecular chaperones or their elimination by AAA+ proteases. Once this first line of defense has been overrun, aggregated proteins are directed to specific compartments, thus protecting the cellular environment from potentially deleterious protein conformations. Organizing protein aggregates might also facilitate the recruitment of protein quality control components, thereby increasing the efficiency of aggregate removal in a subsequent phase. In Saccharomyces cerevisiae application of mild stress (37°C) results, upon inhibiting proteasomal degradation, in partitioning of misfolded proteins between two distinct compartments (Kaganovich, 2008). More mobile misfolded proteins, which are ubiquitylated and likely represent substrates for proteasomal degradation, are sequestered at the JUNQ (juxtanuclear quality control) compartment. Terminally aggregated, insoluble proteins are sorted to the peripheral IPOD (insoluble protein deposit) compartment that also harbors amyloidogenic proteins. To gain further insight into the spatio-temporal organization of misfolded proteins in Saccharomyces cerevisiae, I analyzed the localization of stress-induced protein aggregates by employing various fluorescent reporter proteins that either misfold upon stress application or bind to aggregated proteins. Since little is known about cellular factors involved in the sorting of misfolded proteins, I performed a candidate approach and focused on the Saccharomyces cerevisiae small heat shock proteins (sHsps), namely Hsp26 and Hsp42. I identified Hsp42 as an essential factor in the formation of IPOD-like inclusions. In hsp42 cells misfolded proteins do not accumulate in peripheral inclusions, but seem to be re-directed to the JUNQ. As Hsp42 localizes specifically to IPOD-like inclusions, but is absent from the JUNQ compartment, the lack of peripheral aggregation foci is a direct effect of missing Hsp42, thus illuminating a novel function of sHsps in controlling the cellular sorting of damaged proteins. In contrast, the second Saccharomyces cerevisiae sHsp, Hsp26, does not affect aggregate sorting and is present in both JUNQ and IPOD-like compartments. Transferring the elongated N-terminal domain (NTD) of Hsp42 to Hsp26 enables Hsp26 partially to replace Hsp42 function in aggregate sorting. In contrast, Hsp42 deleted of its NTD is not able to restore the occurrence of peripheral inclusions in hsp42Δ cells. The NTD is thus a key determinant in contributing functional specificity to Hsp42. My data suggest that Hsp42 acts as an adaptor protein that co-aggregates efficiently with misfolded proteins. The sHsp might link such complexes via its NTD to further, so far unknown, sorting factors. Thereby, protein inclusions might be directed to the actin cytoskeleton, which I demonstrate to be crucial for aggregate sorting to JUNQ and IPOD-like compartments. Nonetheless, Hsp42 function is restricted to amorphous aggregates, because the localization of amyloidogenic proteins to IPOD-like inclusions does not depend on Hsp42. Comparing the mobility and stability of aggregated proteins deposited at the JUNQ in wild-type and hsp42Δ cells revealed the JUNQ compartment of hsp42 cells to have a moderate increase in substrate mobility and be solubilized more rapidly by Hsp104. These findings suggest that the Hsp42-dependent sorting to IPOD-like compartments retards substrate resolubilization, thereby potentially reducing substrate load of the quality control system. I also analyzed the spatio-temporal organization of protein aggregates in cells with intact proteasomal degradation during sublethal heat-stress and a subsequent recovery phase allowing for aggregate solubilization. Heat shock generates multiple aggregation foci that are distributed throughout the cell. Sorting of aggregated proteins to JUNQ and IPOD-like deposition sites does not occur upon return to physiological growth conditions. Instead, protein disaggregation takes places in situ and does not require an intact actin cytoskeleton. My data thus demonstrate that the applied stress condition has a profound impact on the organization of misfolded proteins. Moreover, my findings disclose functional divergence of the Saccharomyces cerevisiae sHsps in the refolding and organization of heat shock-generated protein aggregates. Incorporation of Hsp26 facilitates the reactivation of aggregated proteins. In contrast, Hsp42 is not influencing protein refolding, but serves as a sorting factor essential for the persistence of protein inclusions in the cellular periphery.
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570 Biowissenschaften, Biologie
570 Life sciences