scholarly journals PsbQ-Like Protein 3 Functions as an Assembly Factor for the Chloroplast NADH Dehydrogenase-Like Complex in Arabidopsis

2020 ◽  
Vol 61 (7) ◽  
pp. 1252-1261
Author(s):  
Noriko Ishikawa ◽  
Yuki Yokoe ◽  
Taishi Nishimura ◽  
Takeshi Nakano ◽  
Kentaro Ifuku
Keyword(s):  
Nadh Dehydrogenase ◽  
Electron Flow ◽  
Polyacrylamide Gel Electrophoresis ◽  
Oxygen Evolving Complex ◽  
Severe Reduction ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Assembly Factor ◽  
Young Leaves ◽  
Oxygen Evolving ◽  
Blue Native

Abstract Angiosperms have three PsbQ-like (PQL) proteins in addition to the PsbQ subunit of the oxygen-evolving complex of photosystem II. Previous studies have shown that two PQL proteins, PnsL2 and PnsL3, are subunits of the chloroplast NADH dehydrogenase-like (NDH) complex involved in the photosystem I (PSI) cyclic electron flow. In addition, another PsbQ homolog, PQL3, is required for the NDH activity; however, the molecular function of PQL3 has not been elucidated. Here, we show that PQL3 is an assembly factor, particularly for the accumulation of subcomplex B (SubB) of the chloroplast NDH. In the pql3 mutant of Arabidopsis thaliana, the amounts of NDH subunits in SubB, PnsB1 and PsnB4, were decreased, causing a severe reduction in the NDH–PSI supercomplex. Analysis using blue native polyacrylamide gel electrophoresis suggested that the incorporation of PnsL3 into SubB was affected in the pql3 mutant. Unlike other PsbQ homologs, PQL3 was weakly associated with thylakoid membranes and was only partially protected from thermolysin digestion. Consistent with the function as an assembly factor, PQL3 accumulated independently in other NDH mutants, such as pnsl1-3. Furthermore, PQL3 accumulated in young leaves in a manner similar to the accumulation of CRR3, an assembly factor for SubB. These results suggest that PQL3 has developed a distinct function as an assembly factor for the NDH complex during evolution of the PsbQ protein family in angiosperms.

scholarly journals STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes

2015 ◽  
Vol 112 (34) ◽  
pp. E4792-E4801 ◽  
Author(s):  
Guiling Zhao ◽  
Tianyu Li ◽  
Didier X. P. Brochet ◽  
Paul B. Rosenberg ◽  
W. J. Lederer
Keyword(s):  
Cellular Localization ◽  
Ventricular Myocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Heart Cells ◽  
Short Term ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Blue Native ◽  
Cellular Movement

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca2+ sensor, is unclear with respect to its cellular localization, its Ca2+-dependent mobilization, and its action on Ca2+ signaling. Confocal microscopy was used to measure Ca2+ signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca2+ using thapsigargin (2–10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca2+ depletion. Additionally, we found no store-operated Ca2+ entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca2+ content and increased SR Ca2+ leak. These changes in Ca2+ signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca2+ ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca2+ leak and that these actions are independent of store-operated Ca2+ entry, a process that is absent in normal heart cells.

Separation of nuclear protein complexes by blue native polyacrylamide gel electrophoresis

2006 ◽  
Vol 27 (7) ◽  
pp. 1277-1287 ◽  
Author(s):  
Zora Nováková ◽  
Petr Man ◽  
Petr Novák ◽  
Pavel Hozák ◽  
Zdeněk Hodný
Keyword(s):  
Gel Electrophoresis ◽  
Nuclear Protein ◽  
Protein Complexes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Blue Native

scholarly journals Functional Characterization of Reductive Dehalogenases by Using Blue Native Polyacrylamide Gel Electrophoresis

2012 ◽  
Vol 79 (3) ◽  
pp. 974-981 ◽  
Author(s):  
Shuiquan Tang ◽  
Winnie W. M. Chan ◽  
Kelly E. Fletcher ◽  
Jana Seifert ◽  
Xiaoming Liang ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Functional Characterization ◽  
Polyacrylamide Gel ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
Dehalococcoides Mccartyi ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Rdase Genes ◽  
Blue Native

ABSTRACTDehalococcoides mccartyistrains are obligate organohalide-respiring bacteria harboring multiple distinct reductive dehalogenase (RDase) genes within their genomes. A major challenge is to identify substrates for the enzymes encoded by these RDase genes. We demonstrate an approach that involves blue native polyacrylamide gel electrophoresis (BN-PAGE) followed by enzyme activity assays with gel slices and subsequent identification of proteins in gel slices using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). RDase expression was investigated in cultures ofDehalococcoides mccartyistrain BAV1 and in the KB-1 consortium growing on chlorinated ethenes and 1,2-dichloroethane. In cultures of strain BAV1, BvcA was the only RDase detected, revealing that this enzyme catalyzes the dechlorination not only of vinyl chloride, but also of all dichloroethene isomers and 1,2-dichloroethane. In cultures of consortium KB-1, five distinctDehalococcoidesRDases and oneGeobacterRDase were expressed under the conditions tested. Three of the five RDases included orthologs to the previously identified chlorinated ethene-dechlorinating enzymes VcrA, BvcA, and TceA. This study revealed substrate promiscuity for these three enzymes and provides a path forward to further explore the largely unknown RDase protein family.

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