Analysis of Protein Biosynthesis and Translocation in p58+/+ and p58−/− Cells
A general role for p58IPK
in reducing substrate burden via cotranslocational degradation was proposed based on the idea that p58IPK
extracts nascent chains whose translocation is temporarily delayed at the translocon during ER stress. Hence, in the absence of p58IPK
, forward translocation would proceed despite any transient delays, resulting in excessive substrate burden in the ER lumen (Oyadomari et al., 2006
). Therefore, during ER stress, protein translocation into the lumen should proceed at a higher efficiency in p58
−/− cells compared with p58
+/+ cells. We tested this idea directly by measuring the relative rates of protein synthesis and ER translocation during normal and acutely stressed conditions in p58
+/+ and p58
−/− cells (A).
Figure 1. p58−/− cells exhibit a lower ER substrate burden than wild-type cells. (A) Wild-type (top) and p58−/− (bottom) MEFs were treated with varying concentrations of DTT (0.1, 0.3, and 1 mM), TG (0.1, 1, and 10 μM), or (more ...)
Analysis of [35S]methionine incorporation into total cellular proteins during both ER stress (TG or DTT treatments) and non-ER stress (arsenite treatment) showed a dose-dependent inhibition of protein synthesis in p58+/+ and p58−/− cells. Whereas the profile of arsenite-dependent inhibition was similar in both cell types, the knockout cells consistently showed ~40–50% less protein synthesis than wild-type cells during either TG- or DTT-mediated ER stress. This increased translational inhibition in p58−/− cells is consistent with the originally characterized role of p58IPK as an eIF2α kinase inhibitor (in this case, PERK).
To assess the newly proposed role for p58IPK
in cotranslocational degradation (Oyadomari et al., 2006
), we combined pulse labeling with fractionation of cells into cytosolic proteins and N-linked glycoproteins (the latter, a surrogate for the nascent ER-translocated fraction). The aim was to directly quantify the substrate burden on the ER during stress in p58
−/− cells relative to matched p58
+/+ cells. As expected, wild-type cells showed a greater reduction in glycoprotein synthesis than cytosolic protein synthesis during each ER stress condition (B, compare top left with top right). This finding demonstrates that during acute stress, the generation of ER substrates (monitored using the glycoprotein fraction) is reduced more than what might have been expected based on the degree of global translational attenuation alone (monitored using the cytosolic fraction). Thus, the ratio of glycoproteins to cytosolic proteins (a parameter termed GCR) decreases during stress and reflects, at least in part, reduced translocation of substrates into the ER (Kang et al., 2006
). When all of the stress conditions (n = 5) were tabulated together, the average GCR for wild-type cells during acute ER stress was ~70% of the unstressed value (C), suggesting up to ~30% overall reduction in ER protein translocation. Contrary to expectations, the GCR in p58
−/− cells under the same conditions was even lower (~50%) than in p58
+/+ cells. Thus, the overall efficiency of protein influx into the stressed ER of p58
−/− cells is not higher than in p58
+/+ cells, and in fact it seems to be slightly lower.
These results suggest that in the absence of p58IPK
, the ER is not subjected to an increased substrate burden under either normal or stressed conditions. This is seen in at least two ways. First, the absolute amount of glycoproteins generated during ER stress in p58
−/− cells (B, bottom right) is consistently lower than in p58
+/+ cells (B, top right) regardless of the nature or severity of the stressor. Second, even when adjusting for the lower rate of overall protein synthesis during stress in p58
−/− cells, the relative
translocation efficiency (as judged by the GCR) is lower (C). Despite this overall lower substrate burden on the ER lumen during stress, p58
−/− cells are nonetheless more sensitive to ER stress as judged by at least two independent parameters. Not only are the cultured cells and certain tissues more prone to ER protein misfolding (Oyadomari et al., 2006
) but also the stress sensor IRE1 is consistently activated to a slightly greater degree as judged by Xbp1 mRNA splicing (D). Considered together, these results demonstrate that p58
−/− cells are more sensitive to ER stress despite a lower substrate burden
(due to both decreased translation and decreased translocation) than in p58
+/+ cells. Hence, a role for p58IPK
in the reduction of substrate burden on the ER lumen during stress seems unlikely. Instead, the decreased capacity of p58
−/− cells to cope with misfolded substrates that are generated during stress suggests a role for p58IPK
in protein processing or maturation.
p58IPK Has an N-Terminal Signal Sequence for ER Targeting and Translocation
Given that substrate folding and maturation are initiated only upon entry into the ER, the principal lesion in p58
−/− cells would seem to be in the lumenal environment. We therefore reexamined the subcellular localization of p58IPK
, which had previously been proposed to be a peripheral protein of the ER membrane (Yan et al., 2002
; Oyadomari et al., 2006
from all species tested, including human, rodent, nematode, and plant, contains a hydrophobic N-terminal region predicted to be a cleavable ER signal sequence (A; data not shown). In vitro-synthesized p58IPK
was translocated into ER-derived microsomes (as judged by a protease protection assay) with an efficiency of ~50%. This was comparable in efficiency to the translocation of the ER lumenal chaperone calerticulin in this same assay system. Most (but not all) of the in vitro translocated p58IPK
seemed to be processed by signal peptidase; hence, it migrated slightly faster than nontranslocated p58. Translocation was abrogated by deletion of the 31 residues constituting the putative p58IPK
signal sequence (Δs.s./p58), whereas replacement of this region with the signal sequence from the secretory hormone prolactin (Prl) conferred efficient translocation (B).
Figure 2. p58IPK contains a functional N-terminal signal sequence. (A) SignalP3.0 analysis of the murine p58IPK N terminus predicts a likely ER targeting signal (solid line) and cleavage site (broken line). In this analysis, the probability of each residue being (more ...)
Fusion of the predicted p58IPK
signal sequence to an unrelated protein (the mature domain of PrP) was sufficient to mediate PrP translocation as evidenced by both glycosylation and protease protection (Supplemental Figure S1). The translocation efficiency of p58-PrP was within the range of previously characterized signal sequence fusions to PrP (Kim et al., 2002
), being more efficient than the interferon-γ signal and slightly less efficient than the native PrP signal. As expected, fusion of the N-terminal region of the cytosolic protein globin to PrP yielded no translocation. Finally, endogenous cellular p58IPK
from multiple sources comigrated with in vitro-translated Δs.s./p58 rather than with full-length p58IPK
(C; data not shown). Collectively, these data suggested that p58IPK
contains a functional cleavable ER signal sequence.
Consistent with this conclusion, p58IPK
engineered to contain a C-terminal glycosylation site (p58CHO
) was quantitatively modified by an N-linked glycan upon expression in NIH3T3 cells (D). Prl/p58CHO
was identically glycosylated, whereas Δs.s./p58CHO
was not glycosylated (D). The glycans on both p58CHO
were fully sensitive to digestion by endoglycosidase H, whereas those of the plasma membrane-resident transferrin receptor were resistant (E). This result suggests that p58IPK
is retained in the ER, consistent with previous immunofluorescence localization studies (Yan et al., 2002
), proteomic analysis of the secretory pathway (Gilchrist et al., 2006
), and our own observations using green fluorescent protein (GFP)-tagged p58IPK
constructs (data not shown). Based on these analyses with chimeric constructs and exogenously expressed protein, we conclude that p58IPK
can be targeted to and translocated across the ER membrane in vitro and in vivo, where it may be a resident of the ER lumen.
Endogenous p58IPK Is an ER-Resident Lumenal Protein
Analysis of the subcellular localization of endogenous p58IPK in cell lysates by a protease protection assay corroborated the results obtained for the exogenously expressed protein. We observed that p58IPK was protected from proteolytic digestion to an extent comparable with other ER lumenal proteins, including BiP and HSP47, whereas cytosolic proteins and cytosolic domains of membrane proteins were protease accessible (A; data not shown). As expected, all of these proteins became protease accessible in the presence of detergent (A). ER stress did not alter the electrophoretic migration or protease protection of p58IPK (A), suggesting that there is no substantial relocalization of endogenous p58IPK during ER stress.
In support of these conclusions based on experiments in cultured cells, p58IPK
was isolated quantitatively with the rough microsomal fraction, and it was absent in the cytosolic fraction of murine liver homogenates prepared from both nontreated and TM-injected animals (B). p58IPK
and other lumenal proteins could be quantitatively extracted from microsomes by even low concentrations of nondenaturing detergents (including digitonin and Triton X-100), but not by high salt treatment that strips microsomal membranes of the peripherally associated targeting protein SRP54 (C). It should be noted that the alkaline extraction conditions previously used to conclude a peripheral location for p58IPK
(Oyadomari et al., 2006
) also efficiently extract lumenal proteins of ER (Nicchitta and Blobel, 1993
) and are thus not contradictory to our results. As in total cell lysates (A), protease protection analysis of isolated liver microsomes (D and Supplemental Figure S2) revealed that p58IPK
, like known ER lumenal proteins, was fully protected from digestion. By contrast, peripherally associated cytosolic proteins and cytosolic domains of transmembrane proteins were sensitive to digestion (D and Supplemental Figure S2). The same results were obtained from pancreatic microsomes (Supplemental Figure S2). Importantly, the microsomally associated fraction of the cytosolic HSP70 chaperone was quantitatively accessible to protease (D). Therefore, p58IPK
is topologically separated from HSP70 and other cytosolic proteins in liver microsomes under both normal and ER-stressed conditions.
Fractionation of digitonin-solubilized microsomes by velocity sedimentation showed p58IPK migrating exclusively in the low-molecular-weight fractions, along with other ER lumenal proteins such as BiP and PDI (E). In contrast, the SEC61α protein was found in higher molecular weight fractions consistent with its presence in ribosome–translocon complexes (E). Although the microsomal-associated HSP70 fractionated heterogenously (E), we did not observe any fraction that simultaneously contained SEC61α, HSP70, and p58IPK. A similar separation of p58IPK and SEC61α was also observed in microsomes solubilized with Triton X-100 rather than digitonin (Supplemental Figure S2). Furthermore, the proportion of HSP70 that cosedimented with the ribosome-translocon complex (in fractions 5–9) was unchanged in the TM-treated microsomes. This result indicates that a marked stress-dependent recruitment of HSP70 to translocons during ER stress does not occur. Even if some subtle changes in HSP70 recruitment were occurring, it seems unlikely to directly or indirectly involve ER-associated p58IPK, because this factor is located in the lumen and does not cofractionate with the translocon.
The localization of p58IPK
in the ER lumen is inconsistent with its putative association (by cross-linking) with nascent VCAM1 when VCAM1 translocation and degradation are pharmacologically inhibited (Oyadomari et al., 2006
). This discrepancy can be reconciled by our observation that p58IPK
translocation into the ER was itself inhibited by the same agent that inhibits VCAM1 translocation (Supplemental Figure S3). Furthermore, when translocation and proteasomal degradation were inhibited simultaneously, nontranslocated VCAM1 and nontranslocated p58IPK
were both found in detergent-insoluble aggregates that are presumably in the cytosol. Thus, interactions observed under these conditions (when multiple basic cellular processes are pharmacologically perturbed) may not accurately reflect the situation in either normal or ER-stressed cells.
P58IPK Associates with BiP in the ER Lumen
The localization of p58IPK
in the ER lumen () together with its lack of any apparent role in reducing substrate burden during acute stress () raised the issue of how p58IPK
might protect the ER from stress (Oyadomari et al., 2006
). Because DnaJ proteins interact with and regulate the function of HSP70 family members (Cheetham and Caplan, 1998
), we speculated that p58IPK
might associate with the lumenal HSP70 family member BiP, a central component in maintaining homeostasis of the ER folding environment. Indeed, immunoprecipitation of BiP from liver microsomes of either untreated or TM-injected animals copurified p58IPK
, and vice versa, whereas neither protein was precipitated by irrelevant or preimmune sera (A).
Figure 4. p58IPK interacts with BiP in the ER lumen. (A) Liver microsomes from nontreated or TM-injected mice were solubilized in 1% Triton X-100 and immunoprecipitated with antiserum against BiP, an antiserum directed against the C terminus of p58IPK, preimmune (more ...)
This association could be recapitulated in cultured cells using overexpressed HA-tagged p58IPK (B). Importantly, the p58–BiP interaction was also observed with Prl/p58 (whose quantitative localization to the ER lumen was ensured by the highly efficient Prl signal sequence). In contrast, Δs.s./p58 did not coimmunoprecipitate BiP (B), indicating that a spurious interaction after cell lysis did not occur. Identical results were also observed with p58CHO, Prl/p58CHO, and Δs.s./p58CHO (data not shown). A p58IPK construct lacking its J-domain also did not coimmunoprecipitate BiP (C), further supporting the notion that the p58IPK interaction with BiP is specific. These results demonstrate a specific interaction between ER-lumenal p58IPK and BiP in both heterologous and endogenous contexts. Thus, by analogy to other J-domain proteins, p58IPK may function as a cochaperone to facilitate the maturation or metabolism of newly synthesized secretory or membrane proteins.
The potential role for p58IPK as a cochaperone for BiP was supported by our observation that ER lumenal p58IPK can associate with a newly translocated secretory protein. Using in vitro translation, we translocated the secretory protein Prl into ER microsomes, and, after isolation of the microsomes, we tested for Prl association with p58IPK by coimmunoprecipitation (A). Radiolabeled Prl was coimmunoprecipitated with antibodies against p58IPK, PDI, and BiP, but not the β subunit of ER glucosidase II or the HA epitope (an irrelevant antibody control). The specificity of this interaction was further confirmed in two additional ways. First, when total translation extracts were used for the analyses, p58IPK coimmunoprecipitated only translocated Prl, and not the nontranslocated precursor (B). Second, the interaction was substantially diminished if the samples were first treated with SDS for 10 min at 37°C before immunoprecipitation (B). These results suggest that p58IPK, like the chaperones BiP and PDI, can interact with a newly synthesized secretory protein. Whether this interaction is direct or indirect (e.g., through BiP) remains to be determined at present.
Figure 5. p58IPK interacts with a secretory protein in the ER lumen. (A) Preprolactin was synthesized in vitro by using rabbit reticulocyte lysate and pancreatic microsomes. Untranslocated (pPrl) and translocated, signal sequence-cleaved material (Prl) are indicated. (more ...)
P58IPK Overexpression Stimulates PrP Maturation in the ER Lumen
The interaction of p58IPK
with BiP and its ability to coassociate (perhaps indirectly) with a secretory protein in the ER lumen suggested a potential role in either maturation or metabolism of secretory and membrane proteins. Because straightforward assays for Prl maturation were not available, we turned to the PrP. Maturation of this polypeptide requires several posttranslational events, including folding, disulfide bond formation, glycosylation, glycosylphosphatidyl inositol anchor addition, and possibly dimerization. This complexity increases the possibility that subtle changes in the overall processing capacity of the ER might be detected as a change in the rate of PrP maturation, even as an indirect consequence. Previous analyses have shown that upon completion of its maturation in the ER lumen, PrP transits to the Golgi where glycan processing causes both a mobility change and resistance to endoglycosidase H digestion (Kang et al., 2006
). Using the disappearance of core-glycosylated PrP and concomitant appearance of Golgi-modified PrP as markers of maturation in a pulse-chase experiment, we analyzed the consequences of p58IPK
Overexpressed p58 and Prl/p58, but not Δs.s./p58, altered PrP metabolism in two detectable ways. First, there was a subtle (~30%) but consistent decrease in fully glycosylated PrP with a concomitant increase in unglycosylated species at the pulse time point (A). Comparison with in vitro-translated markers showed this unglycosylated band to represent precursor (B), suggesting that it resulted from failed translocation of PrP into the ER lumen. Second, at the chase time points, we observed decreased levels of core-glycosylated PrP relative to higher-molecular-weight glycoforms (A). The assignment of this latter species as mature, post-ER forms of PrP was confirmed by its resistance to endoglycosidase H digestion (A). Thus, p58IPK overexpression in the ER lumen, but not the cytosol, has effects on both PrP translocation into and processing within the ER.
Figure 6. p58IPK overexpression alters PrP metabolism. (A) HeLa cells cotransfected with PrP and the indicated p58IPK constructs were pulse labeled for 10 min and then chased for either 30 or 60 min before immunoprecipitation of PrP. The positions of immature PrP (more ...)
When considered with the observation that p58IPK
interacts with BiP (), we interpreted these effects of p58IPK
overexpression on PrP as follows. First, excess p58IPK
in the ER lumen would interact with and titrate BiP away from other J-domain–containing proteins, including the translocon-associated proteins Sec63 and Mtj1. Because PrP translocation into the ER is dependent on lumenal chaperones to compensate for a weak signal sequence (Kang et al., 2006
), its translocation would be diminished slightly by this titration effect. Second, the shift of BiP from its putative role in translocation at the translocon to a role in protein folding in the ER lumen would be the basis for the improved efficiency of PrP transit to the Golgi. In this view, the greater proportion of BiP (along with the J-domain protein p58IPK
) devoted to posttranslocation folding events would either directly or indirectly facilitate PrP maturation.
Several additional observations supported the above-mentioned model. First, p58IPK
overexpression had essentially no effect on translocation of Prl–PrP (B). Prl–PrP, in which the PrP signal sequence is replaced with the highly efficient Prl signal sequence, exhibits lumenal protein-independent translocation (Kang et al., 2006
) and would therefore not be affected by a shift in BiP functionality. Second, the inhibitory effect on PrP translocation by p58IPK
overexpression in the ER lumen was nearly abolished by deletion of the J-domain (C), a region required for interaction with BiP (C). Third, coexpression of BiP with Prl/p58 also minimized the inhibitory effect on PrP translocation (C), further supporting a titration-based mechanism. And finally, the maturation of Prl–PrP was faster upon overexpression of ER lumenal Prl/p58 compared with cytosolic Δss/p58 (D). Thus, p58IPK
in the ER lumen can stimulate PrP maturation independently of any effect on PrP translocation.
Considered together, these findings argue that p58IPK, via its J-domain–mediated interaction with BiP, facilitates the maturation of newly synthesized proteins in the ER lumen. It should be stressed that at present, we do not know whether p58IPK directly interacts with and influences PrP maturation. Coassociation experiments between p58IPK and PrP as shown for Prl () were hampered by the propensity of immature PrP to aggregate under the native immunoprecipitation conditions used in this assay. Nonetheless, the data support the conclusion that p58IPK overexpression can functionally influence the protein processing capacity of the ER lumen. Although overexpressed p58IPK also caused a selective decrease in PrP translocation, this may not be physiologically relevant, because normally, p58IPK is co-up-regulated with BiP and would therefore not have the titration effect that seems to underlie this phenomenon.
A functional role for p58IPK
as a maturation factor in the ER lumen would explain the increased stress sensitivity of p58
−/− cells (e.g., ) and their decreased capacity to deal with misfolded proteins in certain specialized contexts where the secretory pathway is severely taxed. Two apparent examples of this include pancreatic β cells producing mutant insulin, and hepatocytes pharmacologically perturbed in apolipoprotein B maturation (Oyadomari et al., 2006
). Further work analyzing the functional importance of the p58–BiP interaction as it relates to the folding and maturation of insulin and apolipoprotein B will be needed to test this working hypothesis.
The p58IPK Signal Sequence Allows for Inefficient Translocation during ER Stress
Our localization of p58IPK
to the ER lumen raises the puzzling question of how it is able to regulate the cytosolically disposed kinase activities of PERK and PKR. The simplest explanation is that slight inefficiencies in the translocation of p58IPK
into the ER, either constitutively or selectively under certain conditions, generate sufficient amounts of cytosolic p58IPK
to inhibit these kinases. Consistent with this idea, the p58IPK
signal sequence seems to be less efficient (at least in vitro) than the canonical Prl signal sequence ( and Supplemental Figure S1). Indeed, other slightly inefficient signal sequences have been documented to generate small but detectable cytosolic populations (Levine et al., 2005
) that in some instances can have either physiological (Shaffer et al., 2005
) or pathological consequences (Rane et al., 2004
). We have not been able to reliably detect the presence of endogenous p58IPK
in the cytosol by fractionation or protease protection assays of tissue or cultured cells under either normal or stressed conditions (). This makes a proposed stoichiometric role for this protein on the cytosolic face of the abundantly expressed translocon very unlikely. However, given that the kinases proposed to be inhibited by p58IPK
are very low-abundance proteins, even slight or transient translocational inefficiency might be sufficient to account for this alternative functional activity.
To test whether p58IPK translocation would be capable of such regulation, we used glycosylation as a reporter to compare the ER import of overexpressed p58CHO and Prl/p58CHO during ER stress. As expected by virtue of its highly efficient signal sequence, Prl/p58CHO was constitutively translocated (and thus glycosylated) in both the presence and absence of ER stress (A). In contrast, treatment with 100 nM TG had two effects on p58CHO: signal sequence cleavage was inhibited (quantitatively so by 24 h) and glycosylation of p58CHO was blocked (~50% by 24 h; A). Because the glycosylation acceptor site is located very near the C terminus of p58CHO, the presence of glycosylated but signal sequence-uncleaved chains suggests that the inhibition of signal sequence cleavage exists apart from any potential effect on translocation, because these chains are almost certainly translocated. However, a protease protection assay of hypotonic cell lysates demonstrated that although all of the glycosylated chains were protected from digestion and thus translocated, the unglycosylated chains were completely sensitive, suggesting a cytosolic localization (B). Therefore, the inefficient signal sequence of p58IPK protein is capable, at least in principle, of allowing small populations of the protein to be rerouted to the cytosol during ER stress. Hence, up-regulation of p58IPK during the late phase of the UPR could conceivably simultaneously improve ER folding capacity (via its abundant ER lumenal form) while mitigating translational attenuation by PERK inhibition (via a minor cytosolic form). This may provide a novel mechanism to match substrate synthesis with folding capacity during the recovery phase of the UPR. Future quantitative analysis of p58IPK translocation and localization during different cellular conditions will be required to confirm this possibility.
Figure 7. ER stress reduces the efficiency of p58IPK translocation. (A) NIH 3T3 cells were transfected with p58CHO or Prl/p58CHO and treated with 100 nM TG for the indicated times. Lysates were digested with PNGaseF to resolve N-glycosylated from unmodified chains. (more ...)