Search

This study aimed to identify sites of lysine methylation in Saccharomyces cerevisiae and the associated methyltransferases. Hexapeptide ligand affinity chromatography was used to normalize the abundance levels of proteins in whole cell lysate. MS/MS, in association with antibody-based detection, was then used to identify lysine methylated proteins and the precise sites of modification. Lysine methylation was found on the proteins elongation factor (EF) 1-α, 2, and 3A, as well as ribosomal proteins 40S S18-A/B, 60S L11-A/B, L18-A/B, and L42-A/B. Precise sites were mapped in all cases. Single-gene knockouts of known and putative methyltransferase(s), in association with MS/MS, showed that EF1-α is monomethylated by Efm1 at lysin 30 and dimethylated by See1 at lysine 316. Methyltransferase Rkm1 was found to monomethylate 40S ribosomal protein S18-A/B at lysine 48. Knockout analysis also revealed that putative methyltransferase YBR271W affects the methylation of proteins EF2 and 3A; this was detected by Western blotting and immunodetection. This methyltransferase shows strong interspecies conservation and a tryptophan-containing motif associated with its active site. We suggest that enzyme YBR271W is named EF methyltransferase 2 (Efm2), in line with the recent naming of YHL039W as Efm1. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The large-scale analysis of protein complexes is an emerging challenge in the field of proteomics. Currently, there are few methods available for the fractionation of protein complexes that are compatible with downstream proteomic techniques. Here, we describe the technique of blue native continuous elution electrophoresis (BN-CEE). It combines the features of blue native PAGE (BN-PAGE) and continuous elution electrophoresis (CEE), generating liquid-phase fractions of protein complexes of up to 800 kDa. The resulting complexes can be further analysed by BN-PAGE, by SDS-PAGE and/or by MS. This can help define the constituent proteins of many complexes and their stoichiometry. As BN-CEE is also micropreparative, with a capacity to separate milligram quantities ofprotein complexes, it will assist the study ofproteins oflower abundance. In this regard, the acrylamide concentration and elution rate during separation can be controlled to help 'zoom in' on particular high mass regions and thus complexes of interest. We illustrate the utility of the technique in the analysis of Saccharomyces cerevisiae cellular lysate. © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100 kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH 3 by 80 mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.