Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress
- Cornell University, United States
- Massachusetts Institute of Technology, United States
Figures
Figure 1
BiP contains a conserved cysteine that is dispensable for yeast viability.
(A) Ribbon diagram of human BiP nucleotide binding domain in complex with calcium-ADP (PDB entry 3IUC) (Wisniewska et al., 2010). ADP and the conserved BiP cysteine are shown as colored sticks. …
Figure 2
A cysteine-less BiP strain is sensitive to increased ER oxidation.
(A) CSY170 and CSY278 strains containing an integrated galactose-inducible ERO1* were spotted onto SMM or SMM Gal plates, and plates were incubated for 2 d (glucose) or 3 d (galactose) at 30°C. (B) …
Figure 3
BiP's cysteine is oxidized upon hyperoxidation of the ER by Ero1*.
(A) Schematic for the biotin-switch assay used in panels B and C. (B) The biotin-switch assay was used on lysates prepared from strains CSY316 and CSY319 containing either pCS452 or an empty vector. …
Figure 4
Substitution of the BiP cysteine with an amino acid containing a negatively charged or large side chain enables protection against hyper-oxidation of the ER lumen.
(A and B) CSY278 containing (A) plasmids pCS681, pCS685, pCS802, or empty vector and (B) plasmids pCS681, pCS685, pCS687, pCS688, pCS750, or empty vector were spotted on SMM-leu or SMM Gal-leu …
Figure 5
Replacement of the BiP cysteine with aspartic acid, phenylalanine, tyrosine, or tryptophan results in decreased BiP function.
(A) CSY214 containing the plasmids pCS681, pCS802, pCS687, pCS688, pCS750 or empty vector were spotted onto SMM plates with or without 5-fluoroorotic acid (5-FOA) and incubated for 2 d at 30°C. (B) …
Figure 6
Overexpression of Ero1* causes an accumulation of untranslocated polypeptides.
(A) CSY44 and (B) CSY172 containing plasmids pAF112 (ERO1; E), pCS452 (ERO1*; E*), or empty vector were cultured in galactose medium for 5 hr to induce Ero1 and Suc2 expression. Accumulation of …
Figure 7
A BiP ATPase mutant is not sufficient to protect cells during oxidative stress.
(A) ATP hydrolysis was assessed by determining the fraction of [alpha-32P]ATP converted to [alpha-32P]ADP as described in the 'Materials and methods'. Data represent the means ± SD of three …
Figure 8
BiP cysteine mutants that protect cells during oxidative stress are more effective than wild-type BiP in suppressing polypeptide aggregation.
(A–C) Denatured rhodanese was diluted to a final concentration of 1 µM in the presence of 4 µM BSA or wild-type, mutant, peroxide-treated, or alkylated BiP. Samples in panels B and C were mock …
Tables
Table 1
Plasmids
| Name | Description | Markers | Source |
|---|---|---|---|
| pJC8 | UPRE-LacZ reporter | CEN URA3 LEU2 | Cuozzo and Kaiser, 1999 |
| pCS852 | UPRE-LacZ reporter | CEN URA3 | This study |
| pAF112 | PGAL1-ERO1-myc | CEN URA3 | Sevier et al., 2007 |
| pCS452 | PGAL1-ERO1*-myc | CEN URA3 | Sevier et al., 2007 |
| pCS504 | PGAL1-ero1*-C100A-C105A-myc | CEN URA3 | Sevier et al., 2007 |
| pCS584 | can1::PGAL1-ERO1*-myc | CEN URA3 | This study |
| pCS739 | kar2-C63A | URA3 | This study |
| pCS623 | KAR2 | CEN URA3 | This study |
| pCS681 | KAR2 | CEN LEU2 | This study |
| pCS685 | kar2-C63A | CEN LEU2 | This study |
| pCS802 | kar2-C63D | CEN LEU2 | This study |
| pCS687 | kar2-C63F | CEN LEU2 | This study |
| pCS688 | kar2-C63Y | CEN LEU2 | This study |
| pCS750 | kar2-C63W | CEN LEU2 | This study |
| pCS774 | kar2-T249G | CEN LEU2 | This study |
| pJW5 | kar2-C63D-G445D | CEN LEU2 | This study |
| pCS844 | kar2-C63F-G445D | CEN LEU2 | This study |
| pCS845 | kar2-C63Y-G445D | CEN LEU2 | This study |
| pCS846 | kar2-C63W-G445D | CEN LEU2 | This study |
| pCS757 | KAR2-FLAG | CEN LEU2 | This study |
| pCS760 | kar2-C63A-FLAG | CEN LEU2 | This study |
| pCS630 | kar2-(40-668)-His6 | AMP | This study |
| pCS631 | kar2-(40-668)-C63A-His6 | AMP | This study |
| pJW4 | kar2-(40-668)-C63D-His6 | AMP | This study |
| pCS658 | kar2-(40-668)-C63F-His6 | AMP | This study |
| pCS643 | kar2-(40-668)-C63Y-His6 | AMP | This study |
| pCS644 | kar2-(40-668)-C63W-His6 | AMP | This study |
| pCS639 | kar2-(40-668)-T249G-His6 | AMP | This study |
| pCS675 | GST-sec63J-(121-221) | AMP | This study |
| pCS817 | His6-kar2-(42-682) | KAN | This study |
| pCS818 | His6-kar2-(42-682)-C63A | KAN | This study |
| pCS822 | His6-kar2-(42-682)-C63D | KAN | This study |
| pCS819 | His6-kar2-(42-682)-C63F | KAN | This study |
| pCS820 | His6-kar2-(42-682)-C63Y | KAN | This study |
| pCS821 | His6-kar2-(42-682)-C63W | KAN | This study |
| pKP113 | His6-kar2-(42-682)-T249G | KAN | This study |
Table 2
Yeast strains
| Strain | Genotype | Source |
|---|---|---|
| CKY263/CSY5 | MATa GAL2 ura3-52 leu2-3,112 | Lab collections |
| CKY264/CSY6 | MATα GAL2 ura3-52 leu2-3,112 | Lab collections |
| CKY1026/CSY44 | MATa GAL2 ura3-52 leu2-3,112 ire1Δ::KanMX | Lab collections |
| CSY158 | MATa GAL2 ura3-52 leu2-3,112 ire1Δ::NatMX | This study |
| CSY172 | MATa GAL2 ura3-52 leu2-3,112 ire1Δ::NatMX KanMX:PGAL1-SUC2 | This study |
| CSY275 | MATa GAL2 ura3-52 leu2-3,112 kar2-C63A | This study |
| CSY277 | MATa GAL2 ura3-52 leu2-3,112 kar2-C63A ire1Δ::NatMX | This study |
| CSY170 | MATa GAL2 ura3-52 leu2-3,112 can1::PGAL1-ERO1*-myc | This study |
| CSY278 | MATa GAL2 ura3-52 leu2-3,112 kar2-C63A can1::PGAL1-ERO1*-myc | This study |
| CSY214 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX [pCS623] | This study |
| CSY289 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX [pCS681] | This study |
| CSY290 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX [pCS685] | This study |
| CSY368 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX [pCS802] | This study |
| CSY292 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX [pCS687] | This study |
| CSY293 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX [pCS688] | This study |
| CSY308 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX pep4Δ::NatMX [pCS623] | This study |
| CSY316 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX pep4Δ::NatMX [pCS757] | This study |
| CSY319 | MATa GAL2 ura3-52 leu2-3,112 kar2Δ::KanMX pep4Δ::NatMX [pCS760] | This study |
