Quantitative Characterisation of Low Abundant Yeast Mitochondrial Proteins Reveals Compensation for Haplo-Insufficiency in Different Environments.
Alkisti ManousakiJames BagnallDavid G SpillerLaura Natalia Balarezo-CisnerosMichael WhiteDaniela DelneriPublished in: International journal of molecular sciences (2022)
The quantification of low abundant membrane-binding proteins such as transcriptional factors and chaperones has proven difficult, even with the most sophisticated analytical technologies. Here, we exploit and optimise the non-invasive Fluorescence Correlation Spectroscopy (FCS) for the quantitation of low abundance proteins, and as proof of principle, we choose two interacting proteins involved in the fission of mitochondria in yeast, Fis1p and Mdv1p. In Saccharomyces cerevisiae , the recruitment of Fis1p and Mdv1p to mitochondria is essential for the scission of the organelles and the retention of functional mitochondrial structures in the cell. We use FCS in single GFP-labelled live yeast cells to quantify the protein abundance in homozygote and heterozygote cells and to investigate the impact of the environments on protein copy number, bound/unbound protein state and mobility kinetics. Both proteins were observed to localise predominantly at mitochondrial structures, with the Mdv1p bound state increasing significantly in a strictly respiratory environment. Moreover, a compensatory mechanism that controls Fis1p abundance upon deletion of one allele was observed in Fis1p but not in Mdv1p, suggesting differential regulation of Fis1p and Mdv1p protein expression.
Keyphrases
- saccharomyces cerevisiae
- copy number
- induced apoptosis
- oxidative stress
- high resolution
- cell cycle arrest
- cell death
- mitochondrial dna
- protein protein
- antibiotic resistance genes
- binding protein
- single molecule
- genome wide
- gene expression
- single cell
- dna methylation
- endoplasmic reticulum stress
- mass spectrometry
- cell therapy
- stem cells
- microbial community
- liquid chromatography
- liquid chromatography tandem mass spectrometry
- signaling pathway
- wastewater treatment
- cell proliferation
- solid state
- respiratory tract