Rashel Mizrahi1, Haim Breitbart2. 1. The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel. 2. The Mina & Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel. Electronic address: haim.breitbart@biu.ac.il.
Abstract
BACKGROUND: Mitochondria are the major source of ATP to power sperm motility. Phosphorylation of mitochondrial proteins has been proposed as a major regulatory mechanism for mitochondrial bioenergetics. METHODS: Sperm motility was measured by a computer-assisted analyzer, protein detection by western blotting, membrane potential by tetramethylrhodamine, cellular ATP by luciferase assay and localization of PKA by immuno-electron microscopy. RESULTS: Bicarbonate is essential for the creation of mitochondrial electro-chemical gradient, ATP synthesis and sperm motility. Bicarbonate stimulates PKA-dependent phosphorylation of two 60kDa proteins identified as Tektin and glucose-6-phosphate isomerase. This phosphorylation was inhibited by respiration inhibition and phosphorylation could be restored by glucose in the presence of bicarbonate. However, this effect of glucose cannot be seen when the mitochondrial ATP/ADP exchanger was inhibited indicating that glycolytic-produced ATP is transported into the mitochondria and allows PKA-dependent protein phosphorylation inside the mitochondria. CONCLUSIONS: Bicarbonate activates mitochondrial soluble adenylyl cyclase (sAC) which catalyzes cAMP production leading to the activation of mitochondrial PKA. Glucose can overcome the lack of ATP in the absence of bicarbonate but it cannot affect the mitochondrial sAC/PKA system, therefore the PKA-dependent phosphorylation of the 60kDa proteins does not occur in the absence of bicarbonate. GENERAL SIGNIFICANCE: Production of CO2 in Krebs cycle, which is converted to bicarbonate is essential for sAC/PKA activation leading to mitochondrial membrane potential creation and ATP synthesis.
BACKGROUND: Mitochondria are the major source of ATP to power sperm motility. Phosphorylation of mitochondrial proteins has been proposed as a major regulatory mechanism for mitochondrial bioenergetics. METHODS: Sperm motility was measured by a computer-assisted analyzer, protein detection by western blotting, membrane potential by tetramethylrhodamine, cellular ATP by luciferase assay and localization of PKA by immuno-electron microscopy. RESULTS:Bicarbonate is essential for the creation of mitochondrial electro-chemical gradient, ATP synthesis and sperm motility. Bicarbonate stimulates PKA-dependent phosphorylation of two 60kDa proteins identified as Tektin and glucose-6-phosphate isomerase. This phosphorylation was inhibited by respiration inhibition and phosphorylation could be restored by glucose in the presence of bicarbonate. However, this effect of glucose cannot be seen when the mitochondrial ATP/ADP exchanger was inhibited indicating that glycolytic-produced ATP is transported into the mitochondria and allows PKA-dependent protein phosphorylation inside the mitochondria. CONCLUSIONS:Bicarbonate activates mitochondrial soluble adenylyl cyclase (sAC) which catalyzes cAMP production leading to the activation of mitochondrial PKA. Glucose can overcome the lack of ATP in the absence of bicarbonate but it cannot affect the mitochondrial sAC/PKA system, therefore the PKA-dependent phosphorylation of the 60kDa proteins does not occur in the absence of bicarbonate. GENERAL SIGNIFICANCE: Production of CO2 in Krebs cycle, which is converted to bicarbonate is essential for sAC/PKA activation leading to mitochondrial membrane potential creation and ATP synthesis.
Authors: Kristian R von Schalburg; Brent E Gowen; Jong S Leong; Eric B Rondeau; William S Davidson; Ben F Koop Journal: Histochem Cell Biol Date: 2017-10-05 Impact factor: 4.304
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