Protein complexes of the plant plasma membrane resolved by Blue Native PAGE

Abstract

With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa, several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue‐native polyacrylamide gel electrophoresis (BN‐PAGE). BN‐PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H+‐ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS‐PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low‐abundance protein complexes, e.g. under different physiological conditions.

Involvement of multimeric protein complexes in mediating the capacitation-dependent binding of human spermatozoa to homologous zonae pellucidae

“…To facilitate these studies we have employed blue native polyacrylamide gel electrophoresis (BN-PAGE), an electrophoretic technique originally developed for the analysis of mitochondria (Nijtmans et al, 2002;Schagger and von Jagow, 1991;Wittig et al, 2006), to isolate intact, biologically active protein complexes from human spermatozoa. Although this technique has been widely embraced as a method for isolating membrane bound protein complexes in cells from species as diverse as plants, algae and bacteria (Eubel et al, 2005;Heuberger et al, 2002;Katz et al, 2007;Kjell et al, 2004), this is the first report of BN-PAGE being used for the analysis of human spermatozoa. The results provide novel, compelling evidence that the binding of human spermatozoa to the ZP is orchestrated by multimeric protein complexes.…”

Plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes

“…In common with the cellulose synthases, callose synthases of plants may consist of complexes containing a number of proteins. The GSL proteins have been identified in high‐molecular‐weight complexes on gels (Li et al ., 2003; Kjell et al ., 2004), but other polypeptides were also present in the region containing the GSL proteins. Similarly, several polypeptides are usually present in callose synthase‐enriched fractions, but it remains unclear whether they are integral components of a callose synthase complex.…”

Salt-induced Changes in the Plasma Membrane Proteome of the Halotolerant Alga Dunaliella salina as Revealed by Blue Native Gel Electrophoresis and Nano-LC-MS/MS Analysis

“…To circumvent well known limitations in separation and quantification of membrane proteins by conventional 2D gel electrophoresis, we used blue native (BN)/SDS-PAGE to separate hydrophobic membrane proteins and complexes in their native state. The method was previously applied for separating mitochondrial membrane protein complexes (12), chloroplast protein complexes (13), whole cell lysates (14), and in some preliminary reports on plant plasma membrane (15).…”