Inside mitochondrial respiratory chain, electrons from reduced substrates are passed through electron transport chain (ETC) complexes (I, II, III and IV) to oxygen, forming water and causing protons to be pumped across the mitochondrial inner membrane (Hulbert et al., 2007). This activity of the ETC drives the synthesis of ATP and also generates the electrochemical potential or mitochondrial membrane potential (??m) that is utilized to power the transport of biosynthetic precursors out of the mitochondriaand into the cytoplasm (Jazwinski, 2004). Some of these precursors are the intermediates in the TCA cycle.
When yeast cells (Saccharomyces cerevisiae) are fermenting glucose, the bulk of the ATP is generated at the substrate level through glycolysis. This generated ATP is used for maintaining ??m through the exchange of mitochondrial ADP for ATP with the help of ADP–ATP translocator present in the inner mitochondrial membrane (Jazwinski, 2013). ??m and the processes it supports may play a crucial role in mitochondrial functionality and affects mitochondrial integrity. However, the loss of ??m leads to mitochondrial dysfunction (Jazwinski, 2013; Wright, 2004).
In fact yeast has been used as a single cell eukaryotic model organism for more than 50 years to study the molecular biology of the cell. This model is a rapid and powerful tool that has enabled a better understanding of human biology and many diseases hence it possesses 23% homologous genes to human (Liu et al., 2017). Yeast mitochondrial metabolism gene, AFG3 (YER017C), is an ATPase family gene. Its protein product, Afg3, acts as a component of the m-AAA protease which is basically an ATP-dependent metalloprotease. Afg3 mediates the degradation of misfolded or unassembled inner membrane proteins of mitochondria (Arlt et al., 1996). This protein is necessary for the correct assembly of mitochondrial respiratory chain and ATPase complexes (Arlt et al., 1998; Di Bella et al., 2010). Thus AFG3 is known to play an important role in regulating electron transport chain complexes (Nolden et al., 2005).
AFG3 is homologous to human AFG3L2 and SPG7. In human they interact with each other on the mitochondrial inner membrane to form the m-AAA metalloproteinase complex (Arlt et al., 1996, 1998). The SPG7 gene that is located on human chromosome 16, encodes a protein called paraplegin (Casari et al., 1998; De Michele et al., 1998) and point mutations associated with this gene cause autosomal recessive spastic paraplegia, a neurodegenerative disorder that is characterized by a slow, gradual, progressive weakness and spasticity of the lower limbs. SPG7 mutations have also been associated with other undiagnosed ataxia (Pfeffer et al., 2015; Warnecke et al., 2010). Furthermore, SPG7 deficiency with null mutation is associated with impaired respiratory activities and mitochondrial functions (Warnecke et al., 2010). So yeast AFG3 gene and its deletion mutant afg3? has been used to study the molecular biology of spinocerebellar ataxia type 28 and hereditary spastic paraplegia in yeast model (Arlt et al., 1996, 1998). Delaney et al., 2013 also found that this mutant is unable to use non-fermentable carbon sources indicating an impaired mitochondrial function exists in this mutant.
Indeed, impaired mitochondrial function or, mitochondrial dysfunction is intimately connected to protein aggregation and protein folding disorders (Callegari and Dennerlein, 2018) and mitochondrial defects have recently been shown to lead to genome instability (Veatch et al., 2009) . In response to a mitochondrial perturbation there exists a stress response mechanism that is communicated to the nucleus to increase the expression of mitochondrial associated protein chaperones referred to as the mitochondria-specific unfolded protein response (UPRmt) (Benedetti et al., 2006). Thus mitochondrial unfolded protein response (UPRmt) is considered as mitochondrial proteostasis pathway (Jovaisaite et al., 2014). UPRmt is activated when protein balance in mitochondria is disturbed. This occurs, for instance, upon accumulation of misfolded or unfolded proteins (Haynes & Ron, 2010). This incident then triggers a mitochondria-to-nuclear stress-signal which induces the transcription of nDNA-encoded mitochondrial molecular chaperones, such as mtHSP60 (Durieux et al., 2011).
Houtkooper et al., 2013 reported that mitochondrial ribosomal protein S5 (Mrps5) and other mitochondrial ribosomal proteins as metabolic regulators. Mrp knockdown triggers mitonuclear protein imbalance, reducing mitochondrial respiration and activating the mitochondrial unfolded protein response. UPRmt is also induced induced by mitochondrial stress, subsequently activating a nuclear transcriptional response, inducing the chaperone HSP-60 to restore mitochondrial proteostasis (Haynes & Ron, 2010).
As human spastic paraplegia causing genes AFG3L2 and SPG7 are related to yeast AFG3 that regulates ETC complex and mitochondrial proteostasis, we assumed that the null mutation of AFG3 might affect mitochondrial health and function. So in the present investigation we aimed to test the effect of AFG3 gene deletion on mitochondrial health and activity by analysing mitochondrial membrane potential and mitochondrial unfolded protein response.