Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis.
One major problem concerning the electrophoresis of mitochondria is the heterogeneity of mitochondrial appearance especially under pathological conditions. We show here the use of zone electrophoresis in a free flow electrophoresis device (ZE-FFE) as an analytical sensor to discriminate between different yeast mitochondrial populations. Impairment of the structural properties of the organelles by hyperosmotic stress resulted in broad separation profiles. Conversely untreated mitochondria gave rise to homogeneous populations reflected by sharp separation profiles. Yeast mitochondria with altered respiratory activity accompanied by a different outer membrane proteome composition could be discriminated based on electrophoretic deflection. Proteolysis of the mitochondrial surface proteome and the deletion of a single major protein species of the mitochondrial outer membrane altered the ZE-FFE deflection of these organelles. To demonstrate the usefulness of ZE-FFE for the analysis of mitochondria associated with pathological processes, we analyzed mitochondrial fractions from an apoptotic yeast strain. The cdc48(S565G) strain carries a mutation in the CDC48 gene that is an essential participant in the endoplasmic reticulum-associated protein degradation pathway. Mutant cells accumulate polyubiquitinated proteins in microsomal and mitochondrial extracts. Subsequent ZE-FFE characterization could distinguish a mitochondrial subfraction specifically enriched with polyubiquitinated proteins from the majority of non-affected mitochondria. This result demonstrates that ZE-FFE may give important information on the specific properties of subpopulations of a mitochondrial preparation allowing a further detailed functional analysis.
Zischka
Hans
H
Institutes of Human Genetics and Toxicology, GSF-National Research Center for Environment and Health, 85764 Munich-Neuherberg, Germany. zischka@gsf.de
Braun
Ralf J
RJ
Marantidis
Enrico P
EP
Büringer
Dietmute
D
Bornhövd
Carsten
C
Hauck
Stefanie M
SM
Demmer
Oliver
O
Gloeckner
Christian J
CJ
Reichert
Andreas S
AS
Madeo
Frank
F
Ueffing
Marius
M
D000251:Adenosine Triphosphatases; D018797:Cell Cycle Proteins / Q000235:genetics; D004586:Electrophoresis / Q000295:instrumentation*; D005947:Glucose / Q000737:chemistry; D019344:Lactic Acid / Q000737:chemistry; D008928:Mitochondria / Q000737:chemistry* / Q000648:ultrastructure; D051336:Mitochondrial Membranes / Q000737:chemistry; D024101:Mitochondrial Proteins / Q000032:analysis* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism; D000074405:Valosin Containing Protein
D018797:Cell Cycle Proteins; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D019344:Lactic Acid; D000251:Adenosine Triphosphatases; C000614177:CDC48 protein, S cerevisiae; D000074405:Valosin Containing Protein; D005947:Glucose