scholarly journals The yeast mitophagy receptor Atg32 is ubiquitinated and degraded by the proteasome

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0241576
Author(s):  
Nadine Camougrand ◽  
Pierre Vigié ◽  
Cécile Gonzalez ◽  
Stéphen Manon ◽  
Ingrid Bhatia-Kiššová
Keyword(s):  
Mitochondrial Membrane ◽  
Normal Growth ◽  
Growth Conditions ◽  
Mass Spectrometry Analysis ◽  
Outer Mitochondrial Membrane ◽  
Post Translational Modifications ◽  
Spectrometry Analysis ◽  
Lysine Residues ◽  
The Stability ◽  
Respiratory Conditions

Mitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of mitochondria in cells grown under respiratory conditions. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Post-translational modifications such as phosphorylation are crucial for the execution of mitophagy. In our study we monitor the stability of Atg32 protein in the yeast S. cerevisiae and show that Atg32 is degraded under normal growth conditions, upon starvation or rapamycin treatment. The Atg32 turnover can be prevented by inhibition of the proteasome activity, suggesting that Atg32 is also ubiquitinated. Mass spectrometry analysis of purified Atg32 protein revealed that at least lysine residue in position 282 is ubiquitinated. Interestingly, the replacement of lysine 282 with alanine impaired Atg32 degradation only partially in the course of cell growth, suggesting that additional lysine residues on Atg32 might also be ubiquitinated. Our results provide the foundation to further elucidate the physiological significance of Atg32 turnover and the interplay between mitophagy and the proteasome.

scholarly journals Ubiquitination of the yeast receptor Atg32 modulates mitophagy

2019 ◽  
Author(s):  
Pierre Vigié ◽  
Cécile Gonzalez ◽  
Stephen Manon ◽  
Ingrid Bhatia-Kissova ◽  
Nadine Camougrand
Keyword(s):  
Mass Spectrometry ◽  
Quality Control ◽  
Stationary Phase ◽  
Proteolytic Activity ◽  
Posttranslational Modifications ◽  
Protein Level ◽  
Mitochondrial Membrane ◽  
Mass Spectrometry Analysis ◽  
Outer Mitochondrial Membrane ◽  
Spectrometry Analysis

AbstractMitophagy, the process that degrades mitochondria selectively through autophagy, is involved in the quality control of these organelles. In yeast, the presence of the Atg32 protein on the outer mitochondrial membrane allows for the recognition and targeting of superfluous or damaged mitochondria for degradation. Some posttranslational modifications, such as phosphorylation, are crucial for the execution of the mitophagy process. In our study, we showed that in the stationary phase of growth, and to a lesser extent during starvation, the Atg32 protein level decreases. The fact that a decline in Atg32 level can be prevented by inhibition of the proteolytic activity of proteasome may indicate that Atg32 is also ubiquitylated. In fact, mass spectrometry analysis of purified Atg32 protein showed ubiquitination of lysine residue in position 282. These different patterns of posttranslational modifications of Atg32 could allow cells to control the mitophagy process carefully.

scholarly journals Mass spectrometry analysis of the variants of histone H3 and H4 of soybean and their post-translational modifications

2009 ◽  
Vol 9 (1) ◽  
pp. 98 ◽  
Author(s):  
Tao Wu ◽  
Tiezheng Yuan ◽  
Sau-Na Tsai ◽  
Chunmei Wang ◽  
Sai-Ming Sun ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Histone H3 ◽  
Mass Spectrometry Analysis ◽  
Post Translational Modifications ◽  
Spectrometry Analysis

scholarly journals Proteomic Identification of an Endogenous Synaptic SUMOylome in the Developing Rat Brain

2021 ◽  
Vol 14 ◽  
Author(s):  
Marie Pronot ◽  
Félicie Kieffer ◽  
Anne-Sophie Gay ◽  
Delphine Debayle ◽  
Raphaël Forquet ◽  
...  
Keyword(s):  
Subcellular Fractionation ◽  
Mass Spectrometry Analysis ◽  
Mammalian Brain ◽  
Synaptic Organization ◽  
Post Translational Modifications ◽  
Spectrometry Analysis ◽  
Functional Changes ◽  
Neuronal Communication ◽  
Associated Proteins ◽  
And Function

Synapses are highly specialized structures that interconnect neurons to form functional networks dedicated to neuronal communication. During brain development, synapses undergo activity-dependent rearrangements leading to both structural and functional changes. Many molecular processes are involved in this regulation, including post-translational modifications by the Small Ubiquitin-like MOdifier SUMO. To get a wider view of the panel of endogenous synaptic SUMO-modified proteins in the mammalian brain, we combined subcellular fractionation of rat brains at the post-natal day 14 with denaturing immunoprecipitation using SUMO2/3 antibodies and tandem mass spectrometry analysis. Our screening identified 803 candidate SUMO2/3 targets, which represents about 18% of the synaptic proteome. Our dataset includes neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins as well as vesicular trafficking and cytoskeleton-associated proteins, defining SUMO2/3 as a central regulator of the synaptic organization and function.

scholarly journals Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

2007 ◽  
Vol 130 (2) ◽  
pp. 157-168 ◽  
Author(s):  
David M. Blodgett ◽  
Julie K. De Zutter ◽  
Kara B. Levine ◽  
Pusha Karim ◽  
Anthony Carruthers
Keyword(s):  
Glucose Transport ◽  
Conformational Changes ◽  
Covalent Modification ◽  
Antibody Binding ◽  
Mass Spectrometry Analysis ◽  
Structural Basis ◽  
Ionization Mass ◽  
Spectrometry Analysis ◽  
C Terminus ◽  
Lysine Residues

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7.

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