The bacterial HslVU ATP-dependent protease is a homolog of the eukaryotic 26 S proteasome. HslU ATPase forms a hexameric ring, and HslV peptidase is a dodecamer consisting of two stacked hexameric rings. In HslVU complex, the HslU and HslV central pores are aligned, and the proteolytic active sites are sequestered in an internal chamber of HslV, with access to this chamber restricted to small axial pores. Here we show that the C-terminal tails of HslU play a critical role in the interaction with and activation of HslV peptidase. A synthetic tail peptide of 10 amino acids could replace HslU in supporting the HslV-mediated hydrolysis of unfolded polypeptide substrates such as ␣-casein, as well as of small peptides, suggesting that the HslU C terminus is involved in the opening of the HslV pore for substrate entry. Moreover, deletion of 7 amino acids from the C terminus prevented the ability of HslU to form an HslVU complex with HslV. In addition, deletion of the C-terminal 10 residues prevented the formation of an HslU hexamer, indicating that the C terminus is required for HslU oligomerization. These results suggest that the HslU C-terminal tails act as a molecular switch for the assembly of HslVU complex and the activation of HslV peptidase.

HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. SulA, which is an inhibitor of cell division and has high tendency of aggregation, is degraded by HslVU protease. Here we show that HslU plays a role not only as a regulatory component for the HslV-mediated proteolysis but also as a molecular chaperone. Purified HslU prevented aggregation of SulA in a concentration-dependent fashion. This chaperone activity required oligomerization of HslU subunits, which could be achieved by ATP-binding or in the presence of high HslU protein concentrations. hsl mutation reduced the SulA-mediated inhibition of cell growth and this effect could be reversed upon overproduction of HslU, suggesting that HslU promotes the ability of SulA to block cell growth through its chaperone function. Thus, HslU appears to have two antagonistic functions: one as a chaperone for promotion of the ability of SulA in cell growth inhibition by preventing SulA aggregation and the other as the regulatory component for elimination of SulA by supporting the HslV-mediated degradation. ß

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Bax inhibitor-1 (BI-1) is an evolutionarily conserved protein that protects cells against endoplasmic reticulum (ER) stress while also affecting the ER stress response. In this study, we examined BI-1-induced regulation of the ER stress response as well as the control of the protein over cell death under ER stress. In BI-1-overexpressing cells (BI-1 cells), proteasome activity was similar to that of control cells; however, the lysosomal fraction of BI-1 cells showed sensitivity to degradation of BSA. In addition, areas and polygonal lengths of lysosomes were greater in BI-1 cells than in control cells, as assessed by fluorescence and electron microscopy. In BI-1 cells, lysosomal pH was lower than in control cells and lysosomal vacuolar H ؉ -ATPase(V-ATPase), a proton pump, was activated, suggesting high H ؉ uptake into lysosomes. Even when exposed to ER stress, BI-1 cells maintained high levels of lysosomal activities, including V-ATPase activity. Bafilomycin, a V-ATPase inhibitor, leads to the reversal of BI-1-induced regulation of ER stress response and cell death due to ER stress. In BI-1 knock-out mouse embryo fibroblasts, lysosomal activity and number per cell were relatively lower than in BI-1 wild-type cells. This study suggests that highly maintained lysosomal activity may be one of the mechanisms by which BI-1 exerts its regulatory effects on the ER stress response and cell death.Eukaryotic cells respond to the threat of protein misfolding in the endoplasmic reticulum (ER) 3 by activating either the ER stress response or the unfolded protein response (UPR) (1, 2). The ER stress response consists of pathways that inhibit protein synthesis, up-regulate chaperone proteins, and increase protein degradation activity. Initiation of the ER stress response occurs when the quantity of unfolded proteins exceeds the capacity of chaperone proteins. GRP78 normally binds to the N-terminal ends of the following three transmembrane proteins: RNAdependent protein kinase-like ER kinase, inositol-requiring enzyme 1␣ (IRE1␣), and activating transcription factor 6 (ATF6) (3, 4). Protein kinase-like ER kinase phosphorylates eukaryotic initiation factor 2␣ (eIF2␣) in the regulation of protein translation (5). IRE1 is related to genes involved in the transport of unfolded proteins out of the ER and in their degradation by ER-associated degradation (ERAD) (6). ATF6␣ can also induce chaperone proteins and precedes IRE1␣-mediated production of the ERAD pathway (6). Lysosomal activity is an ERAD II pathway, whereas ERAD I is a proteasome/ubiquitination pathway (7). The ERAD mechanism increases the protein folding capacity by reducing protein folding loads (7, 8), implying that ERAD is a physiological pathway that can regulate ER stress responses (8, 9).Bax inhibitor-1 (BI-1, also known as a "testis-enhanced gene transcript") is an anti-apoptotic protein that inhibits the activation of Bax and its translocation to the mitochondria (10). Functionally, BI-1 affects Ca 2ϩ leakage from the ER, as measured by Ca 2ϩ -sensitive ER-targete...