Translation attenuation by PERK balances ER glycoprotein synthesis with lipid-linked oligosaccharide flux

Abstract

Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc(3)Man(9)GlcNAc(2) (G(3)M(9)Gn(2)) from the lipid-linked oligosaccharide (LLO) glucose(3)mannose(9)N-acetylglucosamine(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) on Ser(51) by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G(3)M(9)Gn(2)-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2alpha Ser(51) phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser(51) kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway.

Figure 1.

Restricted and unrestricted LLO synthesis, and the effect of CHX. (A) Hypothetical high-pressure liquid chromatograms of LLO glycans illustrate efficient (unrestricted) synthesis of G₃M₉Gn₂-P-P-Dol versus accumulation of M_2-7Gn₂-P-P-Dol intermediates under restricted conditions that simulate metabolic dysfunction. The dashed lines indicate the mannosyl residues that vary in the respective intermediates (Helenius and Aebi, 2004). (B) Dermal fibroblasts were treated with the indicated concentrations of CHX for 50 min. Medium with 0.5 mM glucose was used. For the final 20 min, 40 μCi/ml [³H]mannose was added. [³H] LLO glycans were isolated and characterized by HPLC. The positions of standards are shown. Note that addition of CHX converts the pattern of LLO glycans from restricted to unrestricted. For comparison, an experiment is shown with 2 mM DTT added for 30 min instead of CHX.

Figure 2.

PERK's kinase activity is sufficient to rectify LLO biosynthetic defects and aberrant N-linked glycosylation. Fv2E-PERK cells were cultured in the absence (A and C) or presence (B and D) of 0.5 nM AP20187 for 60 min. Incubation with or without AP20187 was then continued for an additional 20 min in medium containing 0.3 mM glucose (causing restricted LLO synthesis), 10% dialyzed FBS, and 40 μCi/ml [³H]mannose. [³H] LLO glycans (A and B) and [³H]N-linked glycans (C and D) were detected by HPLC. The positions of standards are indicated. x and y indicate N-linked glycans assigned the structures M₉Gn₂ and G₁M₉Gn₂, respectively, and thus derived from G₃M₉Gn₂-P-P-Dol rather than undermannosylated LLO intermediates (Shang and Lehrman, 2004c). Because of low [³H] labeling in N-glycan experiments, interference from spurious electronic noise was minimized by subjecting HPLC data to root-mean-square smoothing with PSI-Plot V.8 (Poly Software International).

Figure 3.

Acute ER stress does not alter LLO synthesis in eIF2α^A51/A51 MEFs. MEFs with two normal eIF2α alleles (eIF2α^S51/S51; A–D) or two alleles with Ser⁵¹Ala replacements (eIF2α^A51/A51; E–H) were cultured in the absence (A and E) or presence of 2 mM DTT for 5 min (B and F), 100 nM TG for 10 min (C and G), or 20 μM CHX for 10 min (D and H). Treatments were then continued for an additional 20 min in medium containing 10% dialyzed FCS, 0.3 mM glucose, and 40 μCi/ml [³H]mannose. [³H] LLO glycans were characterized by HPLC, as described for Fig. 2. The causes of the somewhat different patterns of undermannosylated intermediates in untreated eIF2α^S51/S51 and eIF2α^A51/A51 samples in this figure (A and E) and in Fig. 9 are unknown. mRNA samples corresponding to experiments in A–H were tested for splicing of XBP1 mRNA (I). Spliced (XBP1_S), unspliced (XBP1_U), and hybrid (XBP1_H) PCR products are indicated, as well as the sizes (nucleotides) of the XBP1_S and XBP1_U fragments.

Figure 4.

ER st ress in CHO-K1 cells reduces G₃M₉Gn₂-P-P-Dol consumption. CHO-K1 cells (∼90% confluent; duplicate 100-mm dishes for [³H]mannose labeling and duplicate 150-mm dishes for FACE) were incubated with normal medium (10 mM glucose) in the absence or presence of 2 mM DTT (top) or 150 nM TG (bottom) for the indicated times, and then for 20 min (maintaining absence or presence of DTT or TG) in medium with 0.5 mM glucose (unrestricted LLO synthesis). Open symbols: 10 μCi/ml [³H]mannose was included during the final 20 min, and total [³H] LLO was determined. As shown in Fig. S2 C, under comparable conditions the majority of radioactivity was incorporated into G₃M₉Gn₂-P-P-Dol. Closed symbols: no [³H]mannose was included; instead, G₃M₉Gn₂-ANDS was measured by FACE. Data points are averages of duplicates. (insets) FACE gels displaying G₃M₉Gn₂-ANDS from duplicate dishes obtained at the times (minutes) indicated.

Figure 5.

PERK's kinase activity is sufficient to reduce G₃M₉Gn₂-P-P-Dol consumption. (A–C) Control CHO-K1 (left) or Fv2E-PERK (right) cells were treated without (white bars) or with (black bars) 10 nM AP20187 for 1 h, followed by measurements of [³H]leucine incorporation into protein (means of quadruplicates ± the SEM; A), measurements of [³H]mannose incorporation into total LLO (averages of quadruplicates ± the SEM; B), or detection of G₃M₉Gn₂-ANDS by FACE (duplicates; C) in medium with 0.5 mM glucose (unrestricted LLO synthesis under this condition). For A and B, values above the bars are the percentage of incorporation relative to that in the absence of AP20187.

Figure 6.

Hindering PERK expression by RNA interference or gene disruption prevents inhibition of both protein synthesis and LLO consumption by ER stress inducers. Metabolic labeling of HeLa S3 and MEF lines was done with 0.5 mM glucose (unrestricted LLO synthesis conditions; HPLC data not depicted). (A) HeLa S3 cells were subjected to sham transfection, or transfection with siRNA duplexes PERK-A or -B for 5 h. Cells were left untreated (control; open bars) or treated with either 100 nM TG for 30 min (striped bars) or 2 mM DTT for 20 min (shaded bars). Synthesis of protein (with [³H]leucine; left) and LLO (with [³H]mannose; right) were measured as percentages of the untreated controls. Bars represent means (± the SEM) for 7–8 replicates (protein synthesis) or 8–12 replicates (LLO synthesis) done over 4 independent sets of transfections. (inset) RT-PCR analyses of XBP1 mRNA (representative of 4 independent experiments) from a single gel, cropped for alignment with appropriate bars. Spliced (XBP1_S), unspliced (XBP1_U), and hybrid (XBP1_H) PCR products are indicated, with sizes indicated in Fig. 3. (B) MEFs with normal alleles (PERK^+/+; left) or MEFs harboring disrupted PERK alleles (PERK^−/−; right) were treated with TG or DTT, followed by protein and LLO synthesis measurements, as for A. Bars represent averages (± the SEM) of 3–5 or 6–9 replicates for TG and DTT, respectively, encompassing at least 2 independent experiments.

Figure 7.

Pulse-chase analysis of PERK's effect on LLO flux. Fv2E-PERK cells were pulse- labeled with [³H]mannose for 2 min and chased for up to 10 min, as described in Materials and methods. Cells lacked additional treatments (control) or were treated with either 1 nM AP20187 for 1 h or 2 mM DTT for 20 min before pulse labeling, as well as during the pulse (but not during the chase). The 4-min chase was the earliest point at which [³H] LLOs were reliably detected. (A) LLO glycans from cells chased for the indicated times were analyzed by HPLC. The positions of M₅Gn₂ and G₃M₉Gn₂ are shown by the open and closed arrowheads, respectively. (B) Results from A and a second identically performed experiment were combined (all points are means ± the SEM). HPLC peak heights for M₅Gn₂ and G₃M₉Gn₂ were normalized to mannose content, and G₃M₉Gn₂ percentages were calculated. Circles, control; triangles, AP20187; squares, DTT.

Figure 8.

Treatments with ARS and DIA promote synthesis of G₃M₉Gn₂-P-P-Dol and glycosylation of proteins with G₃M₉Gn₂. Dermal fibroblasts were untreated (controls; A and E), or treated with 0.2 mM DIA for 20 min (B and F), 40 μM ARS for 1 h (C and G), or 2 mM DTT for 20 min (D and H). Cells were incubated with medium containing 10% dialyzed FBS and 40 μCi/ml [³H]mannose for 20 min either during (DIA and DTT) or after (ARS) stress treatments. LLO glycans (A–D) and N-linked glycans (E–H) were analyzed by HPLC. The positions of standards are indicated as in Fig. 2.

Figure 9.

Importance of eIF2α-Ser⁵¹ for the actions of cytoplasmic stress inducers on LLO synthesis. eIF2α^S51/S51 (A–E) or eIF2α^A51/A51 (F–J) MEFs (Fig. 3) were left untreated (CON; A and F) or treated with 40 μM ARS for 1 h (B and G), 0.2 mM DIA for 5 min (C and H), 10 μM DIS for 2 h (D and I), or 2 mM DTT for 5 min (E and J). Incubations were continued for 20 min in medium containing the respective agents and 0.3 mM glucose, 10% dialyzed FBS, and 40 μCi/ml [³H]mannose. [³H] LLO glycans were analyzed by HPLC as in Fig. 3.

Figure 10.

Translational balancing by PERK restores correct protein glycosylation. Symbol and font sizes reflect relative amounts of LLO, protein, and hexose. Arrow thicknesses represent relative activity at each step. Glycans from LLO intermediates are indicated by white squares, with extension to G₃M₉Gn₂ indicated by attachment of black squares. (A) Under normal conditions, LLO intermediates are efficiently extended to G₃M₉Gn₂-P-P-Dol, with proper N-glycosylation. (B) Metabolic dysfunction (for example, by limited hexose supply) reduces flux through the LLO pathway, leading to accumulation of LLO intermediates, incorrect protein glycosylation, ER stress, PERK activation, and phosphorylation of eIF2α. (C) Translational balancing requires only moderate translation attenuation by eIF2α-P. This reduces LLO consumption, allowing LLO intermediates to be extended to G₃M₉Gn₂-P-P-Dol even with metabolic dysfunction. Note that fewer glycoproteins are produced than normal, but they are correctly glycosylated with G₃M₉Gn₂.