Mutant fibrinogen cleared from the endoplasmic reticulum via endoplasmic reticulum-associated protein degradation and autophagy: an explanation for liver disease

Abstract

The endoplasmic reticulum (ER) quality control processes recognize and remove aberrant proteins from the secretory pathway. Several variants of the plasma protein fibrinogen are recognized as aberrant and degraded by ER-associated protein degradation (ERAD), thus leading to hypofibrinogenemia. A subset of patients with hypofibrinogenemia exhibit hepatic ER accumulation of the variant fibrinogens and develop liver cirrhosis. One such variant named Aguadilla has a substitution of Arg375 to Trp in the gamma-chain. To understand the cellular mechanisms behind clearance of the aberrant Aguadilla gamma-chain, we expressed the mutant gammaD domain in yeast and found that it was cleared from the ER via ERAD. In addition, we discovered that when ERAD was saturated, aggregated Aguadilla gammaD accumulated within the ER while a soluble form of the polypeptide transited the secretory pathway to the trans-Golgi network where it was targeted to the vacuole for degradation. Examination of Aguadilla gammaD in an autophagy-deficient yeast strain showed stabilization of the aggregated ER form, indicating that these aggregates are normally cleared from the ER via the autophagic pathway. These findings have clinical relevance in the understanding of and treatment for ER storage diseases.

Figure 1

Aguadilla γD degraded in P. pastoris and S. cerevisiae. A: Representative colony-blot immunoassays of single-copy transformants of P. pastoris and high-expression transformants of S. cerevisiae, each expressing wild-type γD (WTγ) or Aguadilla γD (Agγ) or containing a vector control (C). B: A representative pulse-chase immunoprecipitation of single-copy transformants of P. pastoris expressing wild-type γD (WTγ) or Aguadilla γD (Agγ) or containing the vector control (C). Protein standards are noted to the right in kilodaltons. A high molecular weight (◃) and low molecular weight (▸) form of γD are indicated. C: A representative cycloheximide chase immunoblot of a S. cerevisiae wild-type strain expressing either wild-type γD (WTγ) or Aguadilla γD (Agγ) at high levels off the p426Gal1 vector. A graph of the means ± SEM, from at least three experiments is shown with wild-type γD LMW (solid triangles and solid line), wild-type γD HMW (open triangles and solid line), Aguadilla γD LMW (solid circles and dashed line), and Aguadilla γD HMW (open circles and dashed line).

Figure 2

Structural analysis of intracellular forms of γD. A: Reverse phase HPLC profile of P. pastoris lysate solubilized in 8 mol/L urea, 100 mmol/L Tris-HCl, pH 8.0, and 15 mmol/L DTT. B: Western blot of peaks 1 and 2 together with a fibrinogen control (lane C); the mass of the γ-chain of fibrinogen is 48 kd. C: ESI MS tryptic peptide maps of the LMW γD species (top spectrum) and HMW γD species (bottom spectrum). Both maps show the same expected C-terminal QAGDV ion at 489 m/z, indicating normal transcription termination. However, the N-terminal ion of γD (YVVQIHDITGK; 637 m/z) is missing in the HMW map and replaced by an 837 m/z ion composed of the same sequence together with the C-terminal portion of the propeptide (EAEA YVVQIHDITGK). The 890 m/z ion (EEGVSLEK) is unique to the HMW material and is also predicted to originate from the pro peptide.

Figure 3

HMW γD harbors a glycosylated pro sequence. A representative immunoblot of fractions prepared from the S. cerevisiae wild-type strain expressing high levels of wild-type γD as described in Materials and Methods, with visualization of the extracellular (E) secreted material found in the media, the intracellular (I) whole-cell lysates that includes the secreted γD in the periplasm, and a membrane fraction enriched for ER-derived microsomes (M). The microsome fraction was tested for the presence of N-linked core carbohydrates by processing the sample with endoglycosidase H (EndoH) (+) as described in Materials and Methods. Protein standards are noted to the right in kilodaltons. The HMW (◃) and LMW (▸) forms of γD are indicated.

Figure 4

Aguadilla γD degradation is proteasome, vacuole, and autophagy dependent. A: Representative colony-blot immunoassay for both the wild-type isogenic parent (WT) and the proteasome mutant (pre1:2) strains of S. cerevisiae expressing either wild-type γD (WTγ) or Aguadilla γD (Agγ) at low levels off the p426Met25 vector. B: Representative immunoblots from a cycloheximide chase analysis of the wild-type isogenic parent (WT), pep4Δ vacuolar protease-deficient strain, and atg14Δ autophagy-deficient strain of S. cerevisiae expressing Aguadilla γD (Agγ) at high levels off the p426Gal1 vector. The HMW (◃) and LMW (▸) forms of γD are indicated.

Figure 5

Autophagy plays a role in clearing aggregated γD from the ER. A: Cytosol (C) and microsome (M) fractions were prepared from the atg14Δ strain expressing high levels of Aguadilla γD as described in Materials and Methods. The microsome fractions were untreated (−) or treated with trypsin (+) or with trypsin and Triton X-100 (++). Fractions were examined by immunoblot analysis using antisera to the cytosolic marker protein, PGK, fibrinogen (Fib), or the ER luminal chaperone, BiP. B: Representative immunoblots of fractions from a 5% (top, fraction 1) to 60% (bottom, fraction 20) sucrose density gradient separation of microsome lysates prepared from wild-type (WT) and atg14Δ strains expressing high levels of Aguadilla γD. A short exposure (se) of lane 20 is shown to the right of the blots, and densitometry indicates that there is approximately five times more γD aggregate in the atg14Δ strain compared with that found in the wild-type strain.

Figure 6

Proposed model for Aguadilla γD quality control. Aguadilla γD is targeted to ERAD and thus exits the ER by retrotranslocation with subsequent degradation by the proteasome (P). When overexpressed, excess soluble Aguadilla γD (γ) exits the ER by vesicle transport, transits the Golgi (G), and is sorted to the vacuole (V) for degradation; excess Aguadilla γD that aggregates (γγγγ) within the ER is sent to the vacuole via autophagy.