A Two-step Protein Quality Control Pathway for a Misfolded DJ-1 Variant in Fission Yeast

Abstract

A mutation, L166P, in the cytosolic protein, PARK7/DJ-1, causes protein misfolding and is linked to Parkinson disease. Here, we identify the fission yeast protein Sdj1 as the orthologue of DJ-1 and calculate by in silico saturation mutagenesis the effects of point mutants on its structural stability. We also map the degradation pathways for Sdj1-L169P, the fission yeast orthologue of the disease-causing DJ-1 L166P protein. Sdj1-L169P forms inclusions, which are enriched for the Hsp104 disaggregase. Hsp104 and Hsp70-type chaperones are required for efficient degradation of Sdj1-L169P. This also depends on the ribosome-associated E3 ligase Ltn1 and its co-factor Rqc1. Although Hsp104 is absolutely required for proteasomal degradation of Sdj1-L169P aggregates, the degradation of already aggregated Sdj1-L169P occurs independently of Ltn1 and Rqc1. Thus, our data point to soluble Sdj1-L169P being targeted early by Ltn1 and Rqc1. The fraction of Sdj1-L169P that escapes this first inspection then forms aggregates that are subsequently cleared via an Hsp104- and proteasome-dependent pathway.

Keywords: Parkinson disease; Parkinson disease (autosomal recessive, early onset) 7 (PARK7); chaperone; proteasome; proteostasis; ubiquitin.

FIGURE 1.

Sdj1 is a fission yeast homolog of DJ-1 and is induced by stress. A, ClustalW sequence alignment of human (Hs) DJ-1 and S. pombe (Sp) SPAC22E12.03c (Sdj1). Identical and homologous residues have been shaded black and gray, respectively. The secondary structure elements of DJ-1 are indicated by colored bars. Note that both the putative active site cysteine residue Cys-106 (Cys-111 in fission yeast) (marked in red) and the disease relevant Leu-166 (L169 in fission yeast) (marked in green) are conserved. B, GST and GST-tagged Sdj1 were used in pulldown experiments with purified His₆-tagged Sdj1 or Sdj1-L169P. The precipitated material was analyzed by SDS-PAGE and blotting for the His₆-tagged Sdj1 variants (upper panel) or, as a control for even loading, GST (lower panel). C, the plot shows the changes in thermodynamic stability upon all the possible single-site mutations of Sdj1 calculated by FoldX. The value of L169P is marked (green). Note that most Sdj1 mutations are not predicted to affect the Sdj1 structure (values near 0 kcal/mol), very few are predicted to structurally stabilize Sdj1 (negative ΔΔG values), whereas the L169P mutation (green) should destabilize the Sdj1 structure (positive ΔΔG value). The inset shows structure of the Sdj1 homodimer (Protein Data Bank code 4QYT) with the position of Leu-169 (green) marked. D, far-UV circular dichroism spectrum of wild type recombinant Sdj1 purified from E. coli. The maximum at 195 nm, and minimum at 208 nm with the broad shoulder at 222 nm, suggest that the protein is folded and highly α-helical. E, purified wild type Sdj1 and Sdj1-L169P were separated into soluble and insoluble fractions by high-speed centrifugation before the fractions were resolved SDS-PAGE and stained with Coomassie Brilliant Blue (CBB).

FIGURE 2.

Sdj1-L169P aggregates with Hsp104. A, wild type cells expressing Cox4-RFP and Sdj1-YFH (upper panels) or Sdj1-L169P-YFH (lower panels) were fixed and analyzed by DIC microscopy or fluorescence microscopy as indicated. Note that Sdj1 is spread throughout the cell, whereas Sdj1-L169P forms intracellular aggregates. No co-localization with the mitochondrial Cox4 protein was observed. B, wild type cells expressing mCherry-tagged PABP (left panels) or Hsp104 (right panels) and Sdj1-L169P-YFH (lower panels) were fixed and analyzed by DIC microscopy or fluorescence microscopy as indicated. Note that the PABP signal is excluded from Sdj1-L169P aggregates (gray arrowhead), whereas the Hsp104 and Sdj1-L169P signals overlap (white arrowheads).

FIGURE 3.

Sdj1-L169P is degraded by the ubiquitin-proteasome system. A, the steady-state levels of Sdj1-YFH, Sdj1-C111A-YFH, and Sdj1-L169P-YFH were compared by SDS-PAGE and Western blotting using antibodies to His₆ (to detect the YFH tag on the Sdj1 variants) and as a loading control α-tubulin. B, the amount of Sdj1-YFH or Sdj1-L169P-YFH was followed in cultures treated with cycloheximide (CHX) for 5 h. To some cultures, 1 mm of the proteasome inhibitor bortezomib (BZ) was also added. Equal loading was checked using antibodies to tubulin. C, the amount of Sdj1-L169P-YFH was followed in wild type, nas6Δ, and atg1Δ strains treated with cycloheximide. Equal loading was checked using antibodies to tubulin. D, quantification of degradation experiments as in C. Wild type, filled square; nas6Δ, filled circle; atg1Δ, filled diamond. The error bars indicate the S.E. ± mean (n = 4). E, fluorescence micrographs showing the Sdj1-L169P containing aggregates in the indicated genetic backgrounds. Note the larger aggregates in the nas6Δ strain. F, the number of the Sdj1-L169P-YFH aggregate containing cells was determined by fluorescence microscopy for the indicated strains. The error bars indicate the S.E. ± mean (n = 3, ***, p < 0.001, Student's t test). G, the stability of Sdj1-L169P aggregates in the indicated strains was determined by counting the number of cells containing aggregates in cultures treated with cycloheximide. The number of aggregate containing cells at 0 h was normalized to 100%. The error bars indicate the S.E. ± mean (S.E.) (n = 3, ***, p < 0.001, Student's t test). Before normalization the data were: wild type (0 h), 14.38 ± 0.47 (S.E.); wild type (5 h), 3.35 ± 0.18; atg1Δ (0 h), 14.45 ± 1.11; atg1Δ (5 h), 2.91 ± 0.60; wild type + BZ (0 h), 14.38 ± 0.47; wild type + BZ (5 h), 15.32 ± 1.24.

FIGURE 4.

Sdj1-L169P degradation depends on chaperones. A, immunoprecipitates (IP) from wild type S. pombe cells expressing Sdj1-YFH, Sdj1-L169P-YFH, and as a control, YFH. After adjusting the loading, the precipitated material was resolved by SDS-PAGE and analyzed by Western blotting, using antibodies to Hsp70 (upper panel) and His₆ (lower panel). B, left panel, the steady-state level of Sdj1-L169P-YFH in the indicated strains was determined by Western blotting of whole cell lysates. Tubulin served as a loading control. Right panel, bar diagrams showing quantification of Western blots normalized to the wild type control. The error bars indicate the S.E. ± mean (n = 3, *, p < 0.05, Student's t test). C, left panel, the amount of Sdj1-L169P-YFH was followed in the indicated chaperone mutant strains treated with cycloheximide. Equal loading was checked using antibodies to tubulin. Note that Sdj1-L169P is stabilized in the ssa2Δ and hsp104Δ strains (boxed). Right panel, bar diagrams showing quantification of Western blots normalized to the signal at 0 h. The error bars indicate the S.E. ± mean (n = 3). D, fluorescence micrographs showing the Sdj1-L169P containing aggregates in the indicated genetic backgrounds. Note the larger aggregates in the ssa2Δ and hsp104Δ strains. E, the number of the Sdj1-L169P-YFH aggregate containing cells was determined by fluorescence microscopy for the indicated strains. The error bars indicate the S.E. ± mean (n = 3, ***, p < 0.001, Student's t test). DIC, differential interference contrast.

FIGURE 5.

The E3 Ltn1 targets nascent Sdj1-L169P for degradation. A, the steady-state level of Sdj1-L169P-YFH in the indicated strains was determined by Western blotting of whole cell lysates. Tubulin served as a loading control. B, the amount of Sdj1-L169P-YFH was followed in the wild type, ltn1Δ, and rqc1Δ strains treated with cycloheximide (CHX). Equal loading was checked using antibodies to tubulin. C, quantification of degradation experiments as in B. Wild type, filled square; ltn1Δ, filled circle; rqc1Δ, filled diamond. The error bars indicate the S.E. ± mean (n = 4). D, fluorescence micrographs showing the Sdj1-L169P containing aggregates in the indicated genetic backgrounds. Note the larger aggregates in the ltn1Δ and rqc1Δ strains. E, the number of the Sdj1-L169P-YFH aggregate containing cells was determined by fluorescence microscopy for the indicated strains. The error bars indicate the S.E. ± mean (n = 3, *, p > 0.5; ***, p < 0.001, Student's t test). F, the stability of Sdj1-L169P aggregates in the indicated strains was determined by counting the number of cells containing aggregates in cultures treated with cycloheximide. To some cultures (dark gray) the proteasome inhibitor bortezomib (BZ) was also added. The number of aggregate containing cells at 0 h was normalized to 100%. The error bars indicate the S.E. ± mean (n = 3, **, p < 0.01, Student's t test). Before normalization the data were: wild type (0 h), 14.38 ± 0.47 (S.E.); wild type (5 h), 3.35 ± 0.18; wild type + BZ (5 h), 15.32 ± 1.24; ltn1Δ (0 h), 17.77 ± 0.10 (S.E.); ltn1Δ (5 h), 3.52 ± 0.70; ltn1Δ + BZ (5 h), 12.74 ± 0.78; hsp104Δ (0 h), 24.49 ± 1.18; hsp104Δ (5 h), 15.15 ± 1.28; hsp104Δ + BZ (5 h), 15.62 ± 1.56; ltn1Δhsp104Δ (0 h), 23.01 ± 1.79; ltn1Δhsp104Δ (5 h), 11.33 ± 0.19; ltn1Δhsp104Δ + BZ (5 h), 15.54 ± 0.91.

FIGURE 6.

Sdj1-L169P expression is toxic to wild type and hsp104Δ cells. A, growth comparison on solid media of the indicated strains transformed with an nmt1 thiamine-regulated expression construct for Sdj1-L169P. Note that expression of Sdj1-L169P (right panel) inhibits cell growth for the wild type, hsp104Δ, and ltn1Δhsp104Δ strains, but not for the ltn1Δ strain. B, growth comparison on solid media of wild type cells transformed with an nmt1 thiamine-regulated expression construct for Sdj1-L169P either subjected to heat shock prior to plating or untreated. Note that Sdj1-L169P expressing cells grow better when they are heat shocked. C, the steady-state levels of the indicated proteins were determined by blotting of whole cell extracts from wild type, ltn1Δ, hsp104Δ, and ltn1Δhsp104Δ strains. Tubulin served as a loading control.