Fusion of an intact secretory protein permits a misfolded protein to exit from the endoplasmic reticulum in yeast

Figures & data

Fig. 1. CPY*-Inv escaped ERAD but underwent vacuolar degradation.

Note: (A) Schematic drawings of the fusion constructs used in this study. Invertase (Inv) is fused to the carboxyl termini of full-length CPY* and CPY to generate CPY*-Inv and CPY-Inv respectively. All constructs are expressed from the PRC1 promoter on a low-copy plasmid. (B) and (C) CHX chase assays were performed with wild-type (YKS12), hrd1∆ (YKS54), and pep4∆ (YKS28) cells expressing CPY* (B) or CPY*-Inv (C), as described in “Materials and methods.” The remaining proteins were analyzed by immunoblotting with anti-CPY antiserum and anti-3-phosphoglycerate kinase (PGK) antibody. Error bars represent standard deviations for three independent experiments. (D) Protein samples were prepared from the wild-type (YKS12, lanes 1 and 3) or pep4∆ cells (YKS28, lanes 2 and 4) harboring CPY-Inv (lanes 1 and 2) and CPY*-Inv (lanes 3 and 4) plasmid, treated with Endo H, and analyzed by immunoblotting with anti-CPY antiserum. (E) Protein samples were prepared from the pep4∆ cells (YKS28) expressing CPY* (lanes 1 and 2) or CPY*-Inv (lanes 3 and 4) with and without Endo H treatment, and were analyzed by immunoblotting with anti-CPY antiserum. (F) A CHX chase experiment for CPY*-Inv degradation in vps10∆ cells (YKS29) was done as described above.

Fig. 2. CPY*-Inv but not CPY-Inv was sensitive to exogenously added protease.

Note: Cell lysates were prepared from prc1∆hrd1∆ pep4∆ (YKS30) cells expressing CPY (A), CPY* (A), CPY-Inv (B), or CPY*-Inv (B), and then incubated with or without trypsin for the indicated times, as described in “Materials and methods.” After Endo H treatment, samples were analyzed by SDS-PAGE, followed by immunoblot analysis with anti-CPY antiserum. Dots show tryptic digests, and arrowhead indicates the size corresponding to the CPY moiety.

Fig. 3. Blockage of ER exit directed CPY*-Inv to ERAD.

Note: (A) Wild-type (YKS12), sec12-1 (YKS57), and hrd1∆sec12-1 (YKS61) cells containing CPY*-Inv plasmid were grown to early log phase at a permissive temperature. After incubation of the cells at a nonpermissive temperature for 30 min, CHX was added and aliquots were removed at the indicated times for immunoblot analysis, as described in Fig. 1(C). (B) Wild-type (YKS12), sec18-1 (YKS62), and hrd1∆sec18-1 (YKS78) cells containing CPY*-Inv plasmid were analyzed as described above.

Fig. 4. Invertase domain in CPY*-Inv acted as an ER exit signal.

Note: (A) CHX chase experiments were run to monitor the degradation of CPY*-Inv^E486 K in the wild-type (YKS12), hrd1∆ (YKS54), and pep4∆(YKS28) cells, as described in Fig. 1(C). (B) pep4∆ cells (YKS28) expressing CPY*-Inv or CPY*-Inv^E486 K were grown to early log phase, and cell extracts were prepared. Samples were then analyzed by immunoblotting with anti-CPY antiserum.

Fig. 5. CPY*-Inv escaped the ERAD via the p24 complex and Erv29.

Note: (A) CHX chase assays were performed in wild-type (YKS12), emp24∆ (YKS48), emp24∆hrd1∆ (YKS49), and emp24∆pep4∆ cells (YKS50) containing the CPY*-Inv plasmid. (B) CHX chase assays were performed in wild-type (YKS12), erv29∆ (YKS44), erv29∆hrd1∆ (YKS47), and erv29∆pep4∆ (YKS52) cells containing the CPY*-Inv plasmid. (C) CHX chase assays were performed in wild-type (YKS12), erv29∆emp24∆ (YKS59), hrd1∆erv29∆emp24∆ (YKS74), and pep4∆erv29∆emp24∆ cells (YKS75) containing the CPY*-Inv plasmid. (D) pep4∆ (YKS28), pep4∆erv29∆emp24∆ (YKS75), erv29∆pep4∆ (YKS52), and emp24∆pep4∆ cells (YKS50) expressing CPY*-Inv were grown to early log phase, and cell extracts were prepared. After the cell extracts were treated with Endo H or not treated, samples were analyzed by immunoblotting with anti-CPY antiserum.

Fig. 6. The association of Kar2 and Yos9 with CPY*-Inv.

Note: (A) Microsomes were prepared from YTS346 cells expressing CPY-3HA, CPY*-3HA, CPY-Inv, or CPY*-Inv and subjected to native co-immunoprecipitation with anti-HA antibody. Microsomes (Input) and immunoprecipitates (IP: α-HA) were analyzed by immunoblotting with anti-CPY (IB: α-CPY) and anti-Kar2 (IB: α-Kar2) antisera and anti-myc antibody (IB: α-myc). (B) and (C) The relative amounts of Kar2 (B) and Yos9-myc (C) co-precipitated with CPY variants were quantified. The amount of Kar2 or Yos9-myc co-precipitated with CPY* was set to 1. Error bars represent standard deviations for three independent experiments.

Fig. 7. Michaelis–Menten plots relate rates of ER export/ERAD to the concentrations of folded and misfolded secretory proteins.

Note: (A) A simplified FoldEx model for protein export. Unfolded (U) ensembles are converted to the folded or misfolded states (S) via ERAF. The S ensembles bind to ER export and ERAD machineries (E_Ex and E_ERAD, respectively) with specific rate constants (k₁ and k₂). Finally, the S ensembles are incorporated into COPII vesicles (P_Ex) or are retro-translocated to the cytosol (P_ERAD). The relationships between velocity of ER export or ERAD and the concentrations of folded or misfolded secretory proteins in the ER are adapted to Michaelis–Menten kinetics ((B) CPY; (C) CPY*; and (D) CPY*-Inv). Note that the graphs do not reflect actual values from experiments.