Use of (32)P to study dynamics of the mitochondrial phosphoproteome.
Protein phosphorylation is a well-characterized regulatory mechanism in the cytosol, but remains poorly defined in the mitochondrion. In this study, we characterized the use of (32)P-labeling to monitor the turnover of protein phosphorylation in the heart and liver mitochondria matrix. The (32)P labeling technique was compared and contrasted to Phos-tag protein phosphorylation fluorescent stain and 2D isoelectric focusing. Of the 64 proteins identified by MS spectroscopy in the Phos-Tag gels, over 20 proteins were correlated with (32)P labeling. The high sensitivity of (32)P incorporation detected proteins well below the mass spectrometry and even 2D gel protein detection limits. Phosphate-chase experiments revealed both turnover and phosphate associated protein pool size alterations dependent on initial incubation conditions. Extensive weak phosphate/phosphate metabolite interactions were observed using nondisruptive native gels, providing a novel approach to screen for potential allosteric interactions of phosphate metabolites with matrix proteins. We confirmed the phosphate associations in Complexes V and I due to their critical role in oxidative phosphorylation and to validate the 2D methods. These complexes were isolated by immunocapture, after (32)P labeling in the intact mitochondria, and revealed (32)P-incorporation for the alpha, beta, gamma, OSCP, and d subunits in Complex V and the 75, 51, 42, 23, and 13a kDa subunits in Complex I. These results demonstrate that a dynamic and extensive mitochondrial matrix phosphoproteome exists in heart and liver.
Laboratory of Cardiac Energetics and Proteomics Core Facility, National Heart, Lung and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892-1061, USA.
Angel M
AM
Aponte
Darci
D
Phillips
Rachel K
RK
Hopper
D Thor
DT
Johnson
Robert A
RA
Harris
Ksenia
K
Blinova
Emily S
ES
Boja
Stephanie
S
French
Robert S
RS
Balaban
D000818:Animals; D042967:Electron Transport Complex I / Q000737:chemistry / Q000378:metabolism; D007123:Immunoelectrophoresis, Two-Dimensional; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008929:Mitochondria, Heart / Q000737:chemistry / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000737:chemistry / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D010085:Oxidative Phosphorylation; D010750:Phosphoproteins / Q000378:metabolism*; D010761:Phosphorus Radioisotopes / Q000378:metabolism*; D010766:Phosphorylation; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013552:Swine; D013997:Time Factors
D024101:Mitochondrial Proteins; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D020543:Proteome; C021983:complex V (mitochondrial oxidative phosphorylation system); D025261:Mitochondrial Proton-Translocating ATPases; D042967:Electron Transport Complex I