A three-way proteomics strategy allows differential analysis of yeast mitochondrial membrane protein complexes under anaerobic and aerobic conditions.
To investigate the effect of anaerobiosis on the Saccharomyces cerevisiae mitochondrial proteome and the formation of respiratory chain and other protein complexes, we analyzed mitochondrial protein extracts that were enriched from lysates of aerobic and anaerobic steady-state chemostat cultures. We chose an innovative approach in which native mitochondrial membrane protein complexes were separated by 1-D blue native PAGE, which was combined with quantitative analysis of each complex subunit using stable isotope labeling. LC-FT(ICR)-MS/MS analysis was applied to identify and quantify the mitochondrial proteins. In addition, to establish if changes in mitochondrial complex composition occurred under anaerobiosis, we investigated the 1-D blue native PAGE protein migration patterns by Pearson correlation analysis. Surprisingly, we discovered that under anaerobic conditions, where the yeast respiratory chain is not active, the respiratory chain supercomplexes, such as complex V dimer, complex (III)(2)(IV)(2) and complex (III)(2)(IV) were still present, although at reduced levels. Pearson correlation analysis showed that the composition of the mitochondrial complexes was unchanged under aerobic or anaerobic conditions, with the exception of complex II. In addition, this latter approach allowed screening for possible novel complex interaction partners, since for example protein Aim38p, with a yet unknown function, was identified as a possible component of respiratory chain complex IV.
Helbig
Andreas O
AO
Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
de Groot
Marco J L
MJ
van Gestel
Renske A
RA
Mohammed
Shabaz
S
de Hulster
Erik A F
EA
Luttik
Marijke A H
MA
Daran-Lapujade
Pascale
P
Pronk
Jack T
JT
Heck
Albert J R
AJ
Slijper
Monique
M
D000332:Aerobiosis; D000693:Anaerobiosis; D051336:Mitochondrial Membranes / Q000737:chemistry* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism
D024101:Mitochondrial Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins