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. 2003 Aug;14(8):3437-48.
doi: 10.1091/mbc.e02-12-0847. Epub 2003 Apr 17.

Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP

Mehdi Kabani  1 , Stephanie S Kelley, Michael W Morrow, Diana L Montgomery, Renuka Sivendran, Mark D Rose, Lila M Gierasch, Jeffrey L Brodsky
Affiliations

Dependence of endoplasmic reticulum-associated degradation on the peptide binding domain and concentration of BiP

Mehdi Kabani et al. Mol Biol Cell. 2003 Aug.

Abstract

ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453-3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting that the interdomain coupling in Kar2-1p is partially compromised. In contrast, Hsp40 cochaperone-activation of Kar2-1p's ATPase activity was unaffected. Consistent with UPR induction in kar2-1 yeast, an ERAD substrate aggregated in microsomes prepared from this strain but not from wild-type yeast. Overexpression of wild-type BiP increased substrate solubility in microsomes obtained from the mutant, but the ERAD defect was exacerbated, suggesting that simply retaining ERAD substrates in a soluble, retro-translocation-competent conformation is insufficient to support polypeptide transit to the cytoplasm.

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Figures

Figure 1.
Figure 1.
The screen for UPR-induced kar2 mutant yeast (see text for details).
Figure 2.
Figure 2.
Temperature-sensitive phenotypic analysis of the UPR-inducing kar2 mutants. Serial dilutions from log-phase cultures of the wild-type and the indicated mutant strains were inoculated on rich medium and incubated at the indicated temperatures for 3–6 d. The relative strength of UPR activation in the various mutants at 30°C is also indicated; –, no activation; + to +++, varying degrees of UPR activation as determined by the length of time required for the mutant colonies to turn blue in agar-overlay plate assays for β-galactosidase activity (see MATERIALS AND METHODS for details). The nomenclature of the mutants in this figure does not correspond to previously isolated kar2 mutant alleles, but the amino acid position(s) and predicted substitution(s) in the mutant alleles are indicated. Nucleotide substitutions that do not change the encoded amino acid sequence from the wild-type allele (i.e., silent mutations) are not shown. ND, DNA sequence analysis of the mutant allele was not completed.
Figure 3.
Figure 3.
Analysis of ppαF and preBiP translocation in the kar2 mutant strains. Each strain was grown at 26°C to midlog phase and total protein was labeled with 35S-methionine/cysteine for 5 min at either 26° (–) or 37°C (+). Cell extracts were prepared and ppαF and BiP were immunoprecipitated, resolved by SDS-PAGE, and visualized by phosphorimager analysis.
Figure 4.
Figure 4.
The kar2-51 mutant (kar2-1) is translocation-proficient but ERAD-defective in vitro. (A) Microsomes derived from the indicated strains were incubated with in vitro–translated 35S-labeled ΔGppαF (McCracken and Brodsky, 1996) for 40 min at 30°C. Equal amounts of each reaction were either (1) untreated or treated with (2) trypsin or (3) trypsin and Triton X-100, and incubated on ice for 30 min before proteins were TCA-precipitated and resolved by SDS-PAGE and visualized by phosphorimager analysis. The amount of trypsin-protected (translocated) signal sequence-cleaved pαF was 30, 9 and 28% in wild-type, kar2-159, and kar2-51 microsomes, respectively (as averaged from three independent experiments). A representative experiment is shown. (B) 35S-labeled ΔGppαF was translocated into microsomes derived from the indicated strains for 1 h at 20°C, the microsomes were washed, and cytosol and ATP were added to one half of the reactions, whereas the other half was incubated with buffer. After 20 min at 30°C the reactions were quenched with TCA, and precipitated proteins were resolved by SDS-PAGE and phosphorimager analysis to determine the percentage of pαF degraded. The buffer control was set to 100% in each experiment. Data represent the means of three independent experiments (±SD). A representative gel from one experiment is also shown.
Figure 5.
Figure 5.
Mutations in kar2-1 and kar2-133 reside in the peptide-binding domain of BiP. The yeast BiP and E. coli DnaK amino acid sequences were aligned and mutated residues in kar2-1 (P515L) and kar2-133 (T473F) were mapped onto the three-dimensional structure of the DnaK peptide-binding domain (Zhu et al., 1996). The mutated residues are highlighted in yellow, and the approximate location of the lone tryptophan (at position 622) in yeast BiP is indicated in magenta. The bound substrate is displayed in red and is oriented perpendicular to the page.
Figure 6.
Figure 6.
Kar2-1p and Kar2-133p interact functionally with the J domains of Sec63p and Jem1p. Steady state hydrolysis of ATP by wild-type BiP (○), Kar2-1p (•), and Kar2-133p (▵) was measured using 5 μg of BiP and either the (A) GST-Sec63p-J or (B) GST-Jem1p-J fusion proteins. ATPase activity is plotted as nmol of ATP hydrolyzed/min/mg BiP vs. the molar ratio of the J domain–containing fusion proteins to BiP.
Figure 7.
Figure 7.
Kar2-1p and Kar2-133p exhibit reduced peptide affinity and interdomain coupling. (A) Fluorescence anisotropy measurements of F-APPY binding to wild-type BiP (○), Kar2-1p (□), and Kar2-133p (▵), and a best-fit analysis of the data using a single binding site was performed as described by Montgomery et al. (1999). (B) Steady state ATPase assays were performed after assembling reactions on ice in either the presence (filled symbols) or absence (open symbols) of a 1000-fold molar excess of p5: Wild-type BiP (circles), Kar2-1p (squares), and Kar2-133p (triangles).
Figure 8.
Figure 8.
PαF aggregation is enhanced in kar2-1–derived mutant microsomes but can be resolubilized by wild-type BiP. ΔGppαF was translocated into microsomes derived from wild-type (•), kar2-1 (○), and kar2-133 (▴) cells, and kar2-1 yeast transformed with a KAR2 overexpression plasmid (pMR109; □). The microsomes were washed and incubated at 37°C for 30 min before Triton X-100 extraction and sedimentation in a 5–40% sucrose gradient. The percentages of pαF, the in vitro ERAD substrate, in fractions from the top (fraction 1) to the bottom (fraction 14) of the gradient are shown.
Figure 9.
Figure 9.
Increasing the concentration of wild-type BiP exacerbates the kar2-1 ERAD defect and reduces ERAD efficiency in wild-type microsomes. Wild-type (KAR2) and kar2-1 mutant microsomes prepared from strains harboring a control vector (vector) or overexpressing BiP (pMR109) were prepared and an in vitro ERAD assay was performed as described in MATERIALS AND METHODS. Data represent the means of three independent experiments and standard deviations are <10% of the means.
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