Journal: Cellular and Molecular Life Sciences

Article Title: Cold-inducible proteins CIRP and RBM3, a unique couple with activities far beyond the cold

doi: 10.1007/s00018-016-2253-7

Figure Lengend Snippet: Proteins used for alignment in Fig. 1 a

Article Snippet: Bird , Gallus gallus , Gga_CIRP , Q45KQ2 , Aggrecan promoter binding protein (CIRP homologue).

Techniques: Binding Assay, RNA Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: The domain of unknown function 4005 (DUF4005) in an Arabidopsis IQD protein functions in microtubule binding

doi: 10.1016/j.jbc.2021.100849

Figure Lengend Snippet: A conserved region in DUF4005 is necessary for MT binding. A , schematic representations of the mutated versions of ABS6 329–387 used in MT-binding analyses. Red framed boxes indicated the conserved region from P344 to R358. White boxes indicated deleted regions. Δ(P-R), P344–R358, was deleted; Δ(K-R), K354–R358, was deleted; Δ(P-S), P344–S352, was deleted. B , cosedimentation of GST-Δ(P-R), GST-Δ(K-R), and GST-Δ(P-S) with taxol-stabilized MTs in vitro . Cosedimentation assays were performed as in Figure 2 A . GST served as a negative control. C , imaging of GFP fusions of mutated versions of ABS6 329–387 and mRFP-TUB6 in living protoplasts. Δ(P-R)-GFP, Δ(K-R)-GFP, and Δ(P-S)-GFP were transiently expressed in protoplasts from the mRFP-TUB6 line. White arrows pointed to the overlapped signals of Δ(P-S)-GFP and mRFP-TUB6. Bar:10 μm. D , partial sequence alignment of ABS6 DUF4005 and the C-terminal MT-binding region of Kif18A from various species. Sequences were aligned as in Figure 3 B . Kif18A homologs used in the alignment are Danio rerio , NP_956533; Xenopus laevis , XP_018112945; Mus musculus , NP_647464; Gorilla gorilla , XP_004050908; Homo sapiens , NP_112494; Pan paniscus , XP_003830477; Bos taurus , NP_001179838; Balaenoptera musculus , XP_036717440; Sus scrofa , XP_003122951; Anolis carolinensis , XP_003225678; Gallus gallus , NP_001186355.

Article Snippet: Sequences were aligned as in B . Kif18A homologs used in the alignment are Danio rerio , NP_956533; Xenopus laevis , XP_018112945; Mus musculus , NP_647464; Gorilla gorilla , XP_004050908; Homo sapiens , NP_112494; Pan paniscus , XP_003830477; Bos taurus , NP_001179838; Balaenoptera musculus , XP_036717440; Sus scrofa , XP_003122951; Anolis carolinensis , XP_003225678; Gallus gallus , NP_001186355.

Techniques: Binding Assay, In Vitro, Negative Control, Imaging, Sequencing

Journal: Molecular microbiology

Article Title: Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3 -type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

doi: 10.1111/mmi.13321

Figure Lengend Snippet: (A) PccA was purified via a C-terminal Strep-tag and 10 μM of purified proteins were incubated with increasing concentrations of 64CuCl2. Samples were subsequently separated on native PAGE and analyzed by phosphor-imaging. (B) Purified SenC (10μM) was incubated with increasing concentrations of 64CuCl2 with and without prior reduction of the cysteine-containing copper binding motif by TCEP. Samples were then analyzed as in (A). (C) Quantification of the 64Cu binding to SenC (▲), or PccA (◆), or PccAMut, a mutant lacking the two conserved methionine residues of the Cu-binding motif of PCuAC chaperones (■). After 64Cu binding for 10 min at room temperature, samples were purified via Strep-tag and Cu binding was analyzed using a scintillation counter. The data displayed are the mean value of four independent experiments and the error bars represent the standard deviation.

Article Snippet: Recent data have demonstrated that the Sco1 homologue SenC is required for cbb 3 -Cox assembly in R. capsulatus ( Lohmeyer et al. , 2012 ).

Techniques: Purification, Strep-tag, Incubation, Clear Native PAGE, Imaging, Binding Assay, Mutagenesis, Standard Deviation

Journal: Molecular microbiology

Article Title: Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3 -type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

doi: 10.1111/mmi.13321

Figure Lengend Snippet: (A) Spheroplasts of MT1131 cells expressing SenC or from a ΔsenC strain were incubated with Strep-tag purified wild type PccA or PccA-4C, which contained four engineered cysteine residues close to the metal binding site. When indicated, the cysteine-specific crosslinker BMH was added. Samples were subsequently analyzed under non-reducing PAGE, transferred to PVDF membranes and decorated with antibodies against PccA or SenC. PccA-SenC XL indicates the crosslinking product. (B) Spheroplasts were prepared from MT1131 cells expressing either SenC or SenCΔC, which corresponds to a SenC derivative, in which the two cysteine residues of the Cu-binding motif were replaced by alanine. In addition, spheroplasts of the ΔccoGHIS strain expressing SenC were analyzed. In the ΔccoGHIS strain, the cbb3-Cox assembly factors CcoGHIS are absent. The upper panel was decorated with antibodies against SenC and the lower panel with antibodies against PccA.

Article Snippet: Recent data have demonstrated that the Sco1 homologue SenC is required for cbb 3 -Cox assembly in R. capsulatus ( Lohmeyer et al. , 2012 ).

Techniques: Expressing, Incubation, Strep-tag, Purification, Binding Assay

Journal: Molecular microbiology

Article Title: Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3 -type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

doi: 10.1111/mmi.13321

Figure Lengend Snippet: The indicated strains were incubated with para-formaldehyde (PFA) (+) or the reaction buffer only (−). After membrane preparation, cells were solubilized with DDM, and PccAHis was purified via metal affinity chromatography. Purified protein was then separated on SDS-PAGE, transferred to a PVDF membrane and decorated with antibodies against SenC (upper panel) or PccA (lower panel). GK32 carries a ccoNO deletion and lacks cbb3-Cox. In the ΔccoGHIS strain, the cbb3-Cox assembly factors CcoGHIS are absent.

Article Snippet: Recent data have demonstrated that the Sco1 homologue SenC is required for cbb 3 -Cox assembly in R. capsulatus ( Lohmeyer et al. , 2012 ).

Techniques: Incubation, Membrane, Purification, Affinity Chromatography, SDS Page

Journal: Molecular microbiology

Article Title: Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3 -type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

doi: 10.1111/mmi.13321

Figure Lengend Snippet: (A) Total copper concentration (black bars) of MT1131 (wild type), ΔsenC, ΔpccA and ΔsenC-ΔpccA cells was monitored by atomic absorption spectroscopy after growth on MPYE medium or MPYE medium supplemented with 10μM CuSO4 (+) as described in Material and Methods. The manganese and zink concentrations were determined as internal reference. In addition, the copper content of spheroplasts (grey bars) and the supernatants of the spheroplast preparations (periplasmic fraction) (white bars) were also analyzed. The values are the mean of at least three independent experiments and the error bars indicate the SD. Significant p-values (p<0.01) were only observed for the Cu content in the ΔsenC strain (+Cu) compared to the wild type (+Cu). (B) Alkaline phosphatase (PhoA) activity of a ccoA-phoA translational fusion was determined in cell extracts of the indicated strains grown either in the absence of presence of supplementary Cu. The values are the mean of at least two experiments and the error bars indicate the SD.

Article Snippet: Recent data have demonstrated that the Sco1 homologue SenC is required for cbb 3 -Cox assembly in R. capsulatus ( Lohmeyer et al. , 2012 ).

Techniques: Concentration Assay, Atomic Absorption Spectroscopy, Activity Assay

Journal: Molecular microbiology

Article Title: Cooperation between two periplasmic copper chaperones is required for full activity of the cbb 3 -type cytochrome c oxidase and copper homeostasis in Rhodobacter capsulatus

doi: 10.1111/mmi.13321

Figure Lengend Snippet: At low Cu concentrations, the MFS-type Cu transporter CcoA imports Cu2+, which is reduced to Cu+ in the cytoplasm by an unknown mechanism. The P1B-type ATPase CcoI transports Cu+ into the periplasm, where it is bound to PccA, which transfers it to the Sco1-homologue SenC and possibly to other periplasmic Cu-dependent proteins. SenC then delivers Cu+ to the CcoN subunit of cbb3-Cox (red dots indicate the two heme groups in CcoN). In the absence of PccA, Cu+ loading onto SenC and subsequent cbb3-Cox assembly is significantly reduced but still detectable (dashed arrows). At high Cu concentrations, cbb3-Cox assembly proceeds independently of CcoA, SenC and PccA, but still requires CcoI. Although SenC and PccA are dispensable for cbb3-Cox assembly at high Cu concentration, they are very important for regulating Cu homeostasis under these conditions. Absence of SenC leads to increased cytoplasmic Cu concentrations, which return to normal by the simultaneous elimination of PccA. A plausible explanation is the accumulation of Cu-loaded PccA in the absence of SenC, repressing Cu-export via CcoI and leading to increased cytoplasmic Cu pool.

Article Snippet: Recent data have demonstrated that the Sco1 homologue SenC is required for cbb 3 -Cox assembly in R. capsulatus ( Lohmeyer et al. , 2012 ).

Techniques: Concentration Assay