1. Volume 30, Issue 1, Pages 108-113 (April 2008)
Glycolytic Enzyme GAPDH Promotes Peroxide Stress Signaling through Multistep Phosphorelay to a MAPK Cascade 
Susumu Morigasaki, Koichi Shimada, Aminah Ikner, Mitsuaki Yanagida, Kazuhiro Shiozaki 
Molecular Cell 
Volume 30, Issue 1, Pages (April 2008)
DOI: /j.molcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
The H2O2 Stress Signaling Pathway that Activates Spc1 MAPK in S. pombe
See text for details.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2
Tdh1 Forms a Complex with Wis4 MAPKKK and the Mcs4 Response Regulator
(A) Proteins isolated by the affinity purification of Wis4TAP were analyzed by SDS-PAGE and silver staining. The Mcs4 response regulator and the Tdh1 GAPDH were copurified with Wis4TAP (∗). Two additional proteins, translational elongation factor EF-1α (∗∗) and actin (∗∗∗), were identified by mass spectrometry, both of which were also isolated in the control purification from a strain without TAP tag and likely to be nonspecific contaminants.
(B) Specific binding of Tdh1 to Wis4 MAPKKK. Wis4 and unrelated protein kinases, Cmk2 (Sánchez-Piris et al., 2002) and Hal4 (Wang et al., 2005), were expressed with the myc tag in strains expressing Tdh1 with (+) and without (−) a tag of the HA epitope and six histidine residues (HA6H). Cell lysate (top) and proteins isolated by Ni-affinity purification of Tdh1HA6H (bottom) were probed by anti-myc antibodies.
(C) H2O2-induced activation of Spc1 MAPK is compromised in Δtdh1. Exponentially growing cells were treated with H2O2, and the phosphorylated, active form of Spc1 (P-Spc1) and total Spc1 was detected by immunoblotting along the time course.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3
Tdh1 Is Required for the Interaction between Mpr1 HPt Protein and Mcs4 Response Regulator
(A) Wild-type, Δtdh1Δmpr1, and Δmpr1 cells were treated with H2O2, and Spc1 phosphorylation was analyzed as in Figure 2C.
(B) Compromised Mcs4-Mpr1 interaction in tdh1 mutants. Copurification of Mpr1myc with Mcs4HATAP precipitated by IgG beads was tested in tdh1:HA6H (WT), Δtdh1 (Δ), and tdh1-C152S:HA6H (C152S) cell lysate. A tdh1:HA6H mpr1:myc strain expressing untagged Mcs4 was used as a negative control (first lane).
(C) H2O2 stress transiently enhances the interaction of the Mcs4 response regulator with Tdh1 GAPDH. Copurification of FLAG-tagged Mcs4 with Tdh1HA6H precipitated by Ni beads was tested in wild-type and mpr1HQ strains. An mcs4:FLAG strain expressing untagged Tdh1 was used as a negative control (first lane).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Cys-152 Is Essential for the Tdh1 Function in H2O2 Stress Signaling
(A) Transient oxidation of Tdh1 in response to H2O2. Tdh1HA6H was isolated from H2O2-stressed cells, and its oxidized Cys residue was reduced (DTT+) and labeled with iodoacetyl-biotin, followed by detection with peroxidase-conjugated streptavidin. As a control, a parallel labeling experiment was performed without the prior reduction by DTT (DTT−), which resulted in no significant signal. P, a positive control, where all cysteine residues in Tdh1HA6H were labeled; N, a negative control, where all cysteine residues were blocked by iodoacetic acid. Anti-HA immunoblotting shows the Tdh1HA6H protein levels in the samples (lower panel).
(B) Cys-152 is oxidized in Tdh1. Oxidation of wild-type and mutant Tdh1HA6H proteins with a Ser substitution for the individual Cys residues was analyzed before and after 5 min H2O2 stress as in (A).
(C) H2O2 stress does not enhance the interaction of the Tdh1C152S protein with the Mcs4 response regulator. Copurification of Mcs4FLAG with HA6H-tagged Tdh1 or Tdh1C152S precipitated by Ni beads was tested. An mcs4:FLAG strain expressing untagged Tdh1 was used as a negative control (last lane).
(D) H2O2-induced activation of Spc1 MAPK is defective in the tdh1C152S mutant. Phosphorylated (P-Spc1) and total Spc1 (Spc1) in tdh1:HA6H (WT), tdh1C152S:HA6H, and Δtdh1 strains were monitored by immunoblotting after H2O2 stress. Wild-type and mutant Tdh1 proteins in the lysate were detected by anti-HA antibodies (bottom).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions