Abstract
Mitochondrial biogenesis is a highly controlled process that depends on diverse signalling pathways responding to cellular and environmental signals. AMP-activated protein kinase (AMPK) is a critical metabolic enzyme that acts at a central control point in cellular energy homeostasis. Numerous studies have revealed the crucial roles of AMPK in the regulation of mitochondrial biogenesis; however, molecular mechanisms underlying this process are still largely unknown. Previously, we have shown that, in cellular slime mould Dictyostelium discoideum, the overexpression of the catalytic α subunit of AMPK led to enhanced mitochondrial biogenesis, which was accompanied by reduced cell growth and aberrant development. Here, we applied mass spectrometry-based proteomics of Dictyostelium mitochondria to determine the impact of chronically active AMPKα on the phosphorylation state and abundance of mitochondrial proteins and to identify potential protein targets leading to the biogenesis of mitochondria. Our results demonstrate that enhanced mitochondrial biogenesis is associated with variations in the phosphorylation levels and abundance of proteins related to energy metabolism, protein synthesis, transport, inner membrane biogenesis, and cellular signalling. The observed changes are accompanied by elevated mitochondrial respiratory activity in the AMPK overexpression strain. Our work is the first study reporting on the global phosphoproteome profiling of D. discoideum mitochondria and its changes as a response to constitutively active AMPK. We also propose an interplay between the AMPK and mTORC1 signalling pathways in controlling the cellular growth and biogenesis of mitochondria in Dictyostelium as a model organism.
Highlights
To determine whether there is an impact of chronic AMPKα activation on the phosTo determine whether there is an impact of chronic AMPKα activation on the phosphorylation level of mitochondrial proteins in Dictyostelium cells, we first separated total phorylation level of mitochondrial proteins in Dictyostelium cells, we first separated total mitochondrial proteins obtained from vegetative WT and AMPKα-overexpressing cells (HPF444) harvested at the exponential (E) and stationary (S) phases of growth by polyacrylamide gel electrophoresis in SDS (SDS-PAGE) (Figure 2)
To selectively detect mitochondrial phosphoproteins, we stained the gel with Pro-Q Diamond dye, which discriminates mitochondrial proteins obtained from vegetative WT and AMPKα-overexpressing cells 4 of 26 (HPF444) harvested at the exponential (E) and stationary (S) phases of growth by polyacrylamide gel electrophoresis in SDS (SDS-PAGE) (Figure 2)
HPF444 mitochondria; a few protein bands showed either higher or lower and HPF444 mitochondria; a few protein bands showed either higher or lower intensity in the mutant compared to the WT, especially in the mitochondria of exponentially intensity in the mutant compared to the WT, especially in the mitochondria of exponentially growing cells
Summary
Mitochondria are involved in a vast array of biological processes, including energy transformation, calcium signalling, iron–sulphur (Fe-S) cluster biosynthesis, redox balance, the formation of reactive oxygen species (ROS) and the regulation of the apoptotic program, which are fundamental for cell metabolism and survival. The highly flexible nature of mitochondria allows the organelles to respond to cellular needs and environmental signals by changing their morphology, mass, content and dynamics. Mitochondrial biogenesis is a complex and largely controlled process that occurs through the growth and division of existing mitochondria. It requires the increased and coordinated expression of nuclear and mitochondrial genomes, as well as synchronized import and the assembly of proteins and phospholipids in the mitochondrial membranes
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