The CL1 degron used in this study was originally identified in a screen for a genomic sequence that destabilized the cytosolic protein Ura3p by targeting it for degradation via Ubc6p/Ubc7p in the yeast Saccharomyces cerevisiae
(Gilon et al., 1998
). Metzger et al. (2008)
reported that CL1 represents a frame-shifted region of the yeast PMD1
gene and that it contains a strongly hydrophobic region and thus may resemble a misfolded protein when it is exposed. Therefore, a study using a CL1 degron might reveal mechanisms for the targeted removal of improper translational products. A previous study indicated that Ura3p-CL1 degradation in yeast is dependent on the molecular chaperones Ydj1p (a DnaJ homologue) and Ssa1p (Hsp70 homologue) as well as the proteasome (Metzger et al., 2008
). In accordance with the results of that study, we found that the CL1 degron could interact with human homologues of DnaJ, Hsp70, and 26S proteasome subunits in HeLa cells (). Furthermore, we identified BAG-6 as a novel mammalian CL1-associated protein. This observation suggested that BAG-6 may participate in the metabolism of misfolded proteins for proteasomal degradation. Indeed, we found in this study that BAG-6 recognized a CL1 degron substrate and supported its proteasomal degradation. We showed evidence that not only a CL1 model substrate but also newly synthesized polyubiquitinated polypeptides were associated with BAG-6 after proteasome inhibition (). All of our data suggest that BAG-6 provides a transient platform that is necessary for linking the 26S proteasome and its defective substrates for targeted degradation.
It has been reported that ~25% of rapidly degraded polypeptides (RDPs) lose their solubility within an hour of blocking proteasome activity (Qian et al., 2006
). The expression of BAG-6 was diffuse and soluble in normal HeLa cells, whereas treatment with MG132 significantly increased the amount of BAG-6 in the detergent-insoluble fraction (Fig. S5 B). These results support the notion that the BAG-6 protein in the soluble fraction moves to and accumulates in the insoluble aggregates associated with ubiquitinated RDPs during proteasomal dysfunction. It has been reported that polyubiquitinated, aggregation-prone misfolded proteins are transported on microtubules to the MTOC and then form large perinuclear insoluble aggregates/aggresomes (Johnston et al., 1998
; Bence et al., 2001
; Ardley et al., 2003
). Because we found that BAG-6 interacted with polyubiquitinated proteins (; and ) and that a part of BAG-6 moved to the insoluble fractions after treatment with MG132 (Fig. S5 B), we investigated whether BAG-6 colocalized with an aggresome. As shown in Fig. S5 C, confocal microscopic observation revealed that the treatment of MG132 resulted in the formation of a large perinuclear structure and that BAG-6 accumulated on this aggregate. Importantly, we found that BAG-6–positive aggregates were coimmunostained with either polyubiquitin or the intermediate filament protein vimentin, markers for aggresome formation (Johnston et al., 1998
), in the presence of MG132 (Fig. S5 C). Our observations suggest that BAG-6 is functionally linked with the aggresome at the time of proteasome inhibition (Fig. S5 D).
Schubert et al. (2000)
reported that 30% or more of newly synthesized proteins are destroyed by proteasomes of their synthesis. These unstable nascent polypeptides that emerge from the ribosome into the cytosol were designated as defective ribosomal products (Yewdell et al., 1996
). DRiPs are polypeptides that fail to attain a stable conformation because of errors in translation, folding, and post-translational modification as well as errors in transcription and mRNA processing. Puromycin is mistakenly inserted during protein synthesis by the ribosome in place of normal amino acids, resulting in truncated DRiPs containing the drug at their C termini (Vazquez, 1974
; Lelouard et al., 2004
). Our immunocytologic and immunoprecipitation experiments provided evidence that BAG-6 could associate with puromycin-labeled defective nascent polypeptides in vivo. We also showed that BAG-6 controlled in vivo formation of puromycin-induced aggregation structures. Furthermore, our in vitro analysis clearly showed that puromycin-labeled truncated luciferase, a model DRiP substrate, was ubiquitinated and degraded on BAG-6 in rabbit reticulocyte lysates. All of these observations strongly support our conclusion that mammalian BAG-6 is essential for selective elimination of defective nascent chain polypeptides.
It has been shown that newly synthesized proteins are the major source of peptide ligands presented by MHC class I molecules of the major histocompatibility complex on the cell surface (Townsend et al., 1986
; Anton et al., 1997
; Khan et al., 2001
). It has been reported that blocking protein synthesis for 30 min is sufficient to deplete cells of most TAP-transported antigenic peptides (Reits et al., 2000
). Because a similar degree of depletion is reported to be achieved within 15 min of blocking proteasomes, the primary source of peptides is from proteins in the first 15 min of their synthesis (Reits et al., 2000
). To date, there is considerable evidence that a significant source of self and viral peptides is DRiPs, which consist of prematurely terminated and/or misfolded polypeptides (Yewdell et al., 1996
; Yewdell, 2002
; Princiotta et al., 2003
). If BAG-6–mediated processing of DRiPs is a major source of peptide ligands for MHC class I molecules, then blocking BAG-6 function should rapidly decrease the peptide supply and should subsequently slow the cell surface presentation of MHC class I molecules because peptide binding is required for the rapid export of class I molecules. That is the reason why we examined the cell surface presentation of MHC molecules in this study (). Both our FACS and biochemical analyses clearly showed that knockdown of BAG-6 resulted in the suppression of MHC class I cell surface presentation without affecting the intracellular protein transport system. These results also support our hypothesis that BAG-6 is essential for supplying antigenic peptides to the immune system. In good agreement with this observation, we found that BAG-6 was associated with immunoproteasome complexes after treatment with interferon-γ (Fig. S5 A). The initial evidence implicating immunoproteasomes in antigen processing was the discovery that the MHC region contains genes that encode two proteasome subunits and that their expression is controlled by cytokines released by activated T cells (Michalek et al., 1993
; Monaco and Nandi, 1995
; Tanaka and Kasahara, 1998
). When induced, these subunits replace constitutively expressed subunits in newly assembled proteasomes to create immunoproteasomes, which appear to be better at producing peptides favored by MHC class I molecules (Tanaka and Kasahara, 1998
). It is worth recalling that BAG-6 was originally described as an MHC-encoded gene product (Banerji et al., 1990
) and that its expression is apparently enriched in lymphoid tissues (this paper; ). Although we have not yet detected obvious induction of BAG-6 by interferon-γ, we present here an interesting possibility that BAG-6 is a novel factor that modulates immune responses via DRiP-mediated antigen presentations.
The role of BAG-6 in apoptotic cell death appears to be an area of controversy. In several previous studies, BAG-6 has been shown to be required for the induction of apoptosis in response to a variety of stimuli, and the loss of BAG-6 is associated with protection against apoptosis induced by calcium overloading in the ER as well as by menadione and thapsigargin (Desmots et al., 2008
). Currently, we do not exactly know how the reported apoptotic function of BAG-6 is linked to the proteolytic function identified in this study. However, the region that is required for apoptotic control (N-terminal 436 residues in Xenopus
Scythe–BAG-6; Minami et al., 2007
) superficially overlaps with the region of substrate recognition in mammalian BAG-6. In addition, our previous study indicated that the N-terminal region of Scythe–BAG-6 interacts with XEF1AO, a maternal form of polypeptide elongation factor that was suggested to be a potential inducer of apoptosis in vertebrates (Minami et al., 2007
). The binding stimulates polyubiquitin modification and subsequent degradation of XEF1AO in Xenopus
embryos. In addition, we found in this study that BAG-6 provided protection against cell death induced by MG132 and puromycin treatment in mammalian cells. These observations imply that BAG-6–mediated modification of protein degradation is, at least in part, important for apoptotic control caused by the accumulation of aggregation-prone defective proteins. Because aggregated proteins with polyubiquitin have been proposed to be central to the pathology of a number of neurological diseases (Bence et al., 2001
; Taylor et al., 2002
; Ardley et al., 2003
), we are also interested in the possibility that BAG-6 is involved in protein quality control in the neural system.
In the ubiquitin-dependent protein degradation pathway, the substrate sorting process depends on the cooperation of chaperone machineries and ubiquitin chain recognition factors (Hartmann-Petersen and Gordon, 2004
; Verma et al., 2004
; Richly et al., 2005
; Westhoff et al., 2005
). These factors sequentially support the process through protein–protein interactions and thereby escort substrate recognition, ubiquitination, and ubiquitin–protein conjugate presentation to the proteasome (Richly et al., 2005
). Although we do not fully know what kinds of ubiquitin chain recognition factors and ubiquitination machinery are associated with BAG-6 at present, we favor the idea that BAG-6 may possess roles in ubiquitin modification of tethered substrates. We observed that there was an inexplicable increase in BAG-6–bound ubiquitin conjugates for 1 h after MG132 removal (). In our previous study, we reported that Scythe–BAG-6 expression stimulates polyubiquitin modification of XEF1AO substrate (Minami et al., 2007
). These observations suggest that the BAG-6–Scythe complex plays a role in modification of polyubiquitin chains. We suggest that BAG-6 provides a transient platform that links the ubiquitinating machinery, the 26S proteasome, and its newly synthesized substrates to promote their efficient destruction. In any case, we have presented the first evidence that BAG-6–Scythe–BAT3 is a novel polyubiquitinated substrate-associated protein, and our results shed light on the importance of BAG-6 in the degradation of defective proteasomal substrates. Elucidation of the involvement of BAG-6 in the regulation of viral infections and/or onset of neurodegeneration caused by defects in the metabolism of DRiPs should be the next big challenge in understanding the significance of BAG-6 in the development of various human diseases.