A search for peroxisomal membrane protein complexes by BN- PAGE, Mass Spectrometry and Electron Microscopy

In the last decades many of the Pex proteins, involved in protein transport over the peroxisomal membrane, have been genetically and biochemically characterized in detail and models of their interactions have been proposed. However, the models are not based on direct structural studies, such as single particle electron microscopy, which means that there are still many unsolved problems related to these large complexes. We explored the use of BlueNative Gel electrophoresis (BN-PAGE), which is one of the best methods to purify large, transient membrane complexes, and in combination with biochemical characterization, and mass spectrometry looked for ways to assign subunit compositions and interactions. Here, we have performed a pilot study of the major peroxisomal membrane complexes in the yeast species Saccharomyces cerevisiae and Hansenula polymorpha. We have monitored the presence and purification of large, hypothetical Pex complexes by using western blotting with specific antibodies against Pex14p. However, upon 1D and 2D/SDS Blue Native gels electrophoresis from peroxisomal fractions only few protein bands could be attributed to peroxisomes. In fact, no large Pex protein complexes were found, which we also proved by mass spectrometry. This could be related to a very low amount of peroxisomal membrane proteins compared to mitochondrial proteins. Peroxisomes were obtained by differential and sucrose density centrifugation and mitochondria were the major contaminating organelles in purified peak fractions. It appears that further purification of the initial membranes is crucial. This requires more advanced techniques and better separation protocols to separate peroxisomes from mitochondria.