Merck

Mitoflash generated at the Qo site of mitochondrial Complex III.

Journal of cellular physiology (2020-09-16)
Meng Gao, Yuan Qin, Anqi Li, Sailei Wei, Bilin Liu, Xiangang Tian, Guohua Gong
ABSTRACT

The previous research has shown that mitochondrial flash (mitoflash) genesis are functionally and mechanistically integrated with mitochondrial electron transport chain (ETC) energy metabolism. However, the response of mitoflash to superoxide is not entirely consistent with the response of MitoSOX Red. The generation mechanism of mitoflash is still unclear. Here, we investigated mitoflash activities, using the different combinations of ETC substrates and inhibitors, in permeabilized cardiomyocytes or hearts. We found that blocking the complete electron flow, from Complex I to IV, with any one of ETC inhibitors including rotenone (Rot), antimycin A (AntA), myxothiazol (Myxo), stigmatellin, and sodium cyanide, will lead to the abolishment of mitoflashes triggered by substrates in adult permeabilized cardiomyocytes. However, Myxo boosted mitoflashes triggered by the reverse electron of N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate. Moreover, Rot and AntA furtherly enhanced mitoflash activity rather than depressed it, suggesting that mitoflashes generated at the Complex III Qo site. Meanwhile, the inhibition of Complex III protein expression resulted in the activity of Complex III decrease, which decreased mitoflash frequency. The function defect (no change of protein level) of the Qo site of Complex III in aging hearts augmented mitoflash generation confirmed the Qo site function was critical to mitoflash genesis. Thus, our results indicate that mitoflash detected by circularly permuted yellow fluorescent protein is generated at the Qo site of Complex III.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
碳酸氢钠, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
Sigma-Aldrich
透明质酸酶 来源于牛睾丸, Type I-S, lyophilized powder, 400-1000 units/mg solid
Sigma-Aldrich
丙酮酸钠, ReagentPlus®, ≥99%
Sigma-Aldrich
Antimycin A from Streptomyces sp.
Sigma-Aldrich
Adenosine 5′-triphosphate 镁盐, ≥95%, bacterial
Sigma-Aldrich
鱼藤酮, ≥95%
Sigma-Aldrich
皂素, for molecular biology, used as non-ionic surfactant
Sigma-Aldrich
磷酸肌酸 二钠盐 水合物, ≥97%
Sigma-Aldrich
L -肉碱 盐酸盐, synthetic, ≥98%
Sigma-Aldrich
聚-D-赖氨酸 氢溴酸盐, mol wt 30,000-70,000
Sigma-Aldrich
琥珀酸钠 二元 六水合物, ReagentPlus®, ≥99%
Sigma-Aldrich
DL-甘油醛-3-磷酸 溶液, 45-55 mg/mL in H2O
Sigma-Aldrich
腺苷 5′-二磷酸 单钾盐 二水合物, bacterial, ≥95%, powder
Sigma-Aldrich
D -甘露醇, BioXtra, ≥98% (HPLC)
Sigma-Aldrich
L-苹果酸, ≥95% (titration)
Sigma-Aldrich
牛磺酸, suitable for cell culture, meets USP testing specifications
Sigma-Aldrich
牛血清白蛋白 来源于牛血清, chromatographically purified, New Zealand origin, low endotoxin, suitable for cell culture, pH 7, ≥98%
Sigma-Aldrich
四甲基罗丹明甲酯高氯酸盐, ≥95%
Sigma-Aldrich
199 培养基, Modified, with Earle′s salts, without L-glutamine, sodium bicarbonate, and phenol red, powder, suitable for cell culture
Sigma-Aldrich
N,N,N′,N′-四甲基对苯二胺 二盐酸盐, ≥95%, powder
Sigma-Aldrich
粘噻唑, from Myxococcus fulvus Mx f85, ≥98% (HPLC)
Sigma-Aldrich
L -天冬氨酸 钾盐, ≥98% (HPLC)
Sigma-Aldrich
天青B, certified by the Biological Stain Commission
Sigma-Aldrich
二乙胺乙基葡聚糖 盐酸盐, BioReagent, for molecular biology
Sigma-Aldrich
DL-二硫苏糖醇, Vetec, reagent grade, 98%
Sigma-Aldrich
MISSION® esiRNA, targeting human UQCRB