Background: Membrane-associated respiratory complexes, purinosome and many intracellular soluble activities have reported to be organized in dynamic multi-component macromolecular complexes using native PAGE, 2D SDS-PAGE, electron and systematic microscopy and genome-wide GFP fusion library.
Methods: In-gel staining assays, SDS-PAGE and LC-MSMS techniques were performed on cellular extracts to analyze, isolate and identify the proteins associated with glucose 6-phosphate dehydrogenase (G6PDH) and fermentative alcohol dehydrogenase (ADH) I isoform in both Kluyveromyces lactis and Saccharomyces cerevisiae yeasts.
Results: Analysis of LC-MSMS data showed that a large number of components, belonging to glycolysis, pentose phosphate, folding and stress response pathways, were associated with G6PDH and Adh1 putative complexes and that a number of these proteins were identical in either network in both yeasts. However, comparison of in-gel staining assays for hexokinase, phosphoglucoisomerase, acetaldehyde dehydrogenase, ADH and G6PDH showed that, despite their identification in these structures, functional localization of these activities varied according to growth conditions and to NAD(P)+/NAD(P)H redox ratio.
Conclusions: Reported data show that intracellular proteins are organized in large dynamic 'depots' and the NAD(P)+/NAD(P)H redox balance is one of the major factors regulating the assembly and the re-assortment of components inside the different metabolic structures.
General significance: The aim of this work is directed towards the comprehension of the mechanisms involved in the assembly, organization, functioning and dynamic re-assortment of cellular components according to physiological and/or pathological conditions.
Keywords: Depots; In gel-native assay; Mass spectrometry; NAD(P)(+)/NAD(P)H redox balance.
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