Phosphoproteomics of the developing heart identifies PERM1 - An outer mitochondrial membrane protein

https://doi.org/10.1016/j.yjmcc.2021.01.010Get rights and content
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Highlights

  • Proteomes from mouse hearts during early post-natal development cover >10,000 phosphorylation sites on 5000 proteins.

  • Skeletal muscle and heart specific PGC-1- and ERR-induced regulator in muscle 1 (PERM1) expression increases from P2 to P20

  • PERM1 associates with the outer mitochondrial membrane

  • PERM1 is regulated by phosphorylation on its PEST domain by casein kinase 2

  • Ablation of Perm1 alters levels of various lipid species, amino acids, and acylcarnitines

Abstract

Heart development relies on PTMs that control cardiomyocyte proliferation, differentiation and cardiac morphogenesis. We generated a map of phosphorylation sites during the early stages of cardiac postnatal development in mice; we quantified over 10,000 phosphorylation sites and 5000 proteins that were assigned to different pathways. Analysis of mitochondrial proteins led to the identification of PGC-1- and ERR-induced regulator in muscle 1 (PERM1), which is specifically expressed in skeletal muscle and heart tissue and associates with the outer mitochondrial membrane. We demonstrate PERM1 is subject to rapid changes mediated by the UPS through phosphorylation of its PEST motif by casein kinase 2. Ablation of Perm1 in mice results in reduced protein expression of lipin-1 accompanied by accumulation of specific phospholipid species. Isolation of Perm1-deficient mitochondria revealed significant downregulation of mitochondrial transport proteins for amino acids and carnitines, including SLC25A12/13/29/34 and CPT2. Consistently, we observed altered levels of various lipid species, amino acids, and acylcarnitines in Perm1−/− mitochondria. We conclude that the outer mitochondrial membrane protein PERM1 regulates homeostasis of lipid and amino acid metabolites in mitochondria.

Keywords

Heart development
Phosphoproteomics
SILAC
Mitochondria
PERM1
Lipid metabolism

Abbreviations

4EBP1
Eukaryotic initiation factor 4E binding protein 1
ABC
Ammonium bicarbonate
ACN
Acetonitrile
AMD1
S-adenosylmethionine decarboxylase proenzyme 1
BAC
Bacterial artificial chromosomes
BN-PAGE
Blue native polyacrylamide gel electrophorese
CaMKII
Ca2+/calmodulin-dependent protein kinase
CK2
Casein kinase 2
CLPTM1
Cleft lip and palate transmembrane protein 1 homolog
DAG
Diacylglycerol
DGC
Dystrophin glycoprotein complex
DTT
Dithiothreitol
ECM
Extracellular matrix
ER
Endoplasmic reticulum
ETS
Electron transfer system
FASP
Filter-aided sample preparation
FCCP
Trifluoromethoxyphenylhydrazone
fld
fatty liver dystrophic
FSTL1
Follistatin-related protein 1
H&E
Haematoxylin and eosin
IAA
Iodacetamide
IKK
I kappa B kinase
IT
Injection time
LPIN1
Lipin-1
MAM
Mitochondria-associated membrane
MCM
Minichromosome maintenance
MFN2
Mitofusin 2
MKL2
Myocardin-related transcription factor B
NCE
Nominal collisional energy
NMM
Non-mitochondrial membrane
NO
Nitric oxide
PA
Phosphatidic acid
PAP
Phosphatidic acid phosphatase
PE
Phosphatidylethanolamine
PECAM-1
Platelet-endothelial cell adhesion molecule-1
PERM1
Peroxisome proliferator–activated receptor γ coactivator 1- and Estrogen-related receptor–induced regulator in muscle 1
PF4
Platelet factor 4
PI
Phosphatidylinositol
PTM
Post-translational modification
ROX
Residual oxygen consumption
S6K1
Protein S6 kinase
SCX
Strong cation exchange chromatography
SDH
Succinate dehydrogenase
SILAC
Stable isotope labeling with heavy amino acids in cell culture
SLC
Solute carrier family
TBB
4,5,6,7-tetrabromobenzotriazole
TFA
Trifluoroacetic acid
TiO2
Titanium dioxide

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1

These authors contributed equally to this work.