Endoplasmic Reticulum Stress-induced mRNA Splicing Permits Synthesis of Transcription Factor Hac1p/Ern4p That Activates the Unfolded Protein Response

An intracellular signaling from the endoplasmic reticulum (ER) to the nucleus, called the unfolded protein response (UPR), is activated when unfolded proteins are accumulated in the ER under a variety of stress conditions (“ER stress”). We and others recently identified Hac1p/Ern4p as a transcription factor responsible for the UPR in Saccharomyces cerevisiae. It was further reported that Hac1p (238 aa) is detected only in ER-stressed cells, and its expression is mediated by unconventional splicing of HAC1 precursor mRNA. The splicing replaces the C-terminal portion of Hac1p; it was proposed that precursor mRNA is also translated but the putative product of 230 aa is rapidly degraded by the ubiquitin–proteasome pathway. We have identified and characterized the same regulated splicing and confirmed its essential features. Contrary to the above proposal, however, we find that the 238-aa product of mature mRNA and the 230-aa-type protein tested are highly unstable with little or no difference in stability. Furthermore, we demonstrate that the absence of Hac1p in unstressed cells is due to the lack of translation of precursor mRNA. We conclude that Hac1p is synthesized as the result of ER stress-induced mRNA splicing, leading to activation of the UPR.

Mammalian Transcription Factor ATF6 Is Synthesized as a Transmembrane Protein and Activated by Proteolysis in Response to Endoplasmic Reticulum Stress

The unfolded protein response (UPR) controls the levels of molecular chaperones and enzymes involved in protein folding in the endoplasmic reticulum (ER). We recently isolated ATF6 as a candidate for mammalian UPR-specific transcription factor. We report here that ATF6 constitutively expressed as a 90-kDa protein (p90ATF6) is directly converted to a 50-kDa protein (p50ATF6) in ER-stressed cells. Furthermore, we showed that the most important consequence of this conversion was altered subcellular localization; p90ATF6 is embedded in the ER, whereas p50ATF6 is a nuclear protein. p90ATF6 is a type II transmembrane glycoprotein with a hydrophobic stretch in the middle of the molecule. Thus, the N-terminal half containing a basic leucine zipper motif is oriented facing the cytoplasm. Full-length ATF6 as well as its C-terminal deletion mutant carrying the transmembrane domain is localized in the ER when transfected. In contrast, mutant ATF6 representing the cytoplasmic region translocates into the nucleus and activates transcription of the endogenous GRP78/BiP gene. We propose that ER stress-induced proteolysis of membrane-bound p90ATF6 releases soluble p50ATF6, leading to induced transcription in the nucleus. Unlike yeast UPR, mammalian UPR appears to use a system similar to that reported for cholesterol homeostasis.

Regulation of the dolichol pathway in human fibroblasts by the endoplasmic reticulum unfolded protein response

Accumulation of unfolded proteins within the endoplasmic reticulum (ER) of eukaryotic cells triggers the unfolded protein response (UPR), which activates transcription of several genes encoding ER chaperones and folding enzymes. This study reports that conversion of dolichol-linked Man2–5GlcNAc2 intermediates into mature Glc3Man9GlcNAc2 oligosaccharides in primary human adult dermal fibroblasts is also stimulated by the UPR. This stimulation was not evident in several immortal cell lines and did not require a cytoplasmic stress response. Inhibition of dolichol-linked Glc3Man9GlcNAc2 synthesis by glucose deprivation could be counteracted by the UPR, improving the transfer of Glc3Man9GlcNAc2 to asparagine residues on nascent polypeptides. Glycosidic processing of asparagine-linked Glc3Man9GlcNAc2 in the ER leads to the production of monoglucosylated oligosaccharides that promote interaction with the lectin chaperones calreticulin and calnexin. Thus, control of the dolichol-linked Glc3Man9GlcNAc2 supply gives the UPR the potential to maintain efficient protein folding in the ER without new synthesis of chaperones or folding enzymes.

The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

The Cytoplasmic Chaperone Hsp104 Is Required for Conformational Repair of Heat-denatured Proteins in the Yeast Endoplasmic Reticulum

Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24°C are preconditioned at 37°C, proteins denatured by subsequent exposure to 48–50°C can be renatured when the cells are allowed to recover at 24°C. Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult. Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p. We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104. A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated. During recovery at 24°C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional. Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

Specific interaction of the yeast cis-Golgi syntaxin Sed5p and the coat protein complex II component Sec24p of endoplasmic reticulum-derived transport vesicles

The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p/Sec24p; Sec13p/Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, we found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p. Sec24p/Sed5p interaction in vitro was not dependent on the presence of [Sar1p⋅GTP]. The binding of Sec24p to Sed5p is specific; none of the other seven yeast syntaxins bound to this COPII component. Whereas the interaction site of Sec23p is within the N-terminal half of the 926-aa-long Sec24p (amino acid residues 56–549), Sed5p binds to the N- and C-terminal halves of the protein. Destruction by mutagenesis of a potential zinc finger within the N-terminal half of Sec24p led to a nonfunctional protein that was still able to bind Sec23p and Sed5p. Sec24p/Sed5p binding might be relevant for cargo selection during transport-vesicle formation and/or for vesicle targeting to the cis-Golgi.

ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis thaliana

To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [γ-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.

Rer1p as common machinery for the endoplasmic reticulum localization of membrane proteins

Rer1p, a Golgi membrane protein, is required for the correct localization of an endoplasmic reticulum (ER) membrane protein, Sec12p, by a retrieval mechanism from the cis-Golgi to the ER. To test whether or not the role of Rer1p is common to multiple ER membrane proteins, we examined the localization of two other ER membrane proteins, Sec71p and Sec63p, in the wild-type and rer1 mutant yeast cells, using their fusions with an α-mating factor precursor (Mfα1p). Although Sec71p and Sec63p have completely different topology from Sec12p, their Mfα1p fusion proteins were also mislocalized to the trans-Golgi in the rer1 mutant. Overexpression of these fusions caused their mislocalization to the trans-Golgi even in the wild-type cells, and this mislocalization was partially suppressed by the co-overexpression of Rer1p. Either Sec71p or an artificial chimeric protein whose ER localization depends on Rer1p gave a competitive effect on the localization of the Mfα1-Sec71p fusion, which was abolished in rer1. Thus, Rer1p appears to be one of the common limiting components in the retrieval machinery for ER membrane proteins. The results also suggest that Sec71p and Sec63p depend on ER-Golgi recycling, at least partly, for ER localization. We also examined the effect of a mutation in α-COP, a subunit of yeast coatomer, on the localization of these ER membrane proteins. The Mfα1p fusions of Sec12p, Sec71p, and Sec63p were all more or less mislocalized in ret1–1. These observations imply that the roles of Rer1p and coatomer are much more general than thought before.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells

Spectrin (βIΣ∗) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Endoplasmic reticulum quality control of asialoglycoprotein receptor H2a involves a determinant for retention and not retrieval

The human asialoglycoprotein receptor H2a subunit contains a charged pentapeptide, EGHRG, in its ectodomain that is the only sequence absent from the H2b alternatively spliced variant. H2b exits the endoplasmic reticulum (ER) even when singly expressed, whereas H2a gives rise to a cleaved soluble secreted ectodomain fragment; uncleaved membrane-bound H2a molecules are completely retained and degraded in the ER. We have inserted the H2a pentapeptide into the sequence of the H1 subunit (H1i5), which caused complete ER retention but, unexpectedly, no degradation. This suggests that the pentapeptide is a determinant for ER retention not colocalizing in H2a with the determinant for degradation. The state of sugar chain processing and the ER localization of H1i5, which was unchanged at 15°C or after treatment with nocodazole, indicate ER retention and not retrieval from the cis-Golgi or the intermediate compartment. H1i5 folded similarly to H1, and both associated to calnexin. However, whereas H1 dissociated with a half time of 45 min, H1i5 remained bound to the chaperone for prolonged periods. The correct global folding of H2a and H1i5 and of other normal precursors and unassembled proteins and the true ER retention, and not exit and retrieval, suggest a difference in their quality control mechanism compared with that of misfolded proteins, which does involve retrieval. However, both pathways may involve calnexin.

Conversion of cysteine to formylglycine: A protein modification in the endoplasmic reticulum

In sulfatases a Cα-formylglycine residue is found at a position where their cDNA sequences predict a cysteine residue. In multiple sulfatase deficiency, an inherited lysosomal storage disorder, catalytically inactive sulfatases are synthesized which retain the cysteine residue, indicating that the Cα-formylglycine residue is required for sulfatase activity. Using in vitro translation in the absence or presence of transport competent microsomes we found that newly synthesized sulfatase polypeptides carry a cysteine residue and that the oxidation of its thiol group to an aldehyde is catalyzed in the endoplasmic reticulum. A linear sequence of 16 residues surrounding the Cys-69 in arylsulfatase A is sufficient to direct the oxidation. This novel protein modification occurs after or at a late stage of cotranslational protein translocation into the endoplasmic reticulum when the polypeptide is not yet folded to its native structure.

Mobility of cytochrome P450 in the endoplasmic reticulum membrane

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 × 10−10 cm2/s. A coefficient only slightly larger (7.1 × 10−10 cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

Detection of Transient In Vivo Interactions between Substrate and Transporter during Protein Translocation into the Endoplasmic Reticulum

The split-ubiquitin technique was used to detect transient protein interactions in living cells. Nub, the N-terminal half of ubiquitin (Ub), was fused to Sec62p, a component of the protein translocation machinery in the endoplasmic reticulum of Saccharomyces cerevisiae. Cub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution of a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of Cub, serve as a gauge of proximity between the two test proteins linked to Nub and Cub. Using this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-α-factor in vivo. This proximity is confined to the nascent polypeptide chain immediately following the signal sequence. In addition, the extent of proximity depends on the nature of the signal sequence. Cub fusions that bore the signal sequence of invertase resulted in a much lower Ub reconstitution with Nub-Sec62p than otherwise identical test proteins bearing the signal sequence of prepro-α-factor. An inactive derivative of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence-binding complex.