Yuko Iwafune

Electrophoretic analysis of phosphorylation of the yeast 20S proteasome.

The 26S proteasome complex, consisting of two multisubunit complexes, a 20S proteasome and a pair of 19S regulatory particles, plays a major role in the nonlysosomal degradation of intracellular proteins. The 20S proteasome was purified from yeast and separated by two‐dimensional gel electrophoresis (2‐DE). A total of 18 spots separated by 2‐DE were identified as the 20S proteasome subunits by peptide mass fingerprinting with matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry (MALDI‐TOF‐MS). The α2‐, α4‐ and α7‐subunits gave multiple spots, which converged into one spot for each subunit when treated with alkaline phosphatase. The difference of pI between phosphorylated and dephosphorylated spots and their reaction against anti‐phosphotyrosine antibody suggested that the α2‐ and α4‐subunits are phosphorylated either at Ser or at Thr residue, and the α7‐subunit is phosphorylated at Tyr residue(s). These phosphorylated subunits were analyzed by electrospray ionization‐quadrupole time of flight‐tandem MS (ESI‐QTOF‐MS/MS) to deduce the phosphorylation sites. The 20S proteasome has three different protease activities: chymotrypsin‐like, trypsin‐like and peptidylglutamyl peptide‐hydrolyzing activities. The phosphatase treatment increased Km value for chymotrypsin‐like activity of the 20S proteasome, indicating that phosphorylation may play an important role in regulating the proteasome activity.

Co- and post-translational modifications of the 26S proteasome in yeast

The yeast (Saccharomyces cerevisiae) 26S proteasome consists of the 19S regulatory particle (19S RP) and 20S proteasome subunits. We detected comprehensively co‐ and post‐translational modifications of these subunits using proteomic techniques. First, using MS/MS, we investigated the N‐terminal modifications of three 19S RP subunits, Rpt1, Rpn13, and Rpn15, which had been unclear, and found that the N‐terminus of Rpt1 is not modified, whereas that of Rpn13 and Rpn15 is acetylated. Second, we identified a total of 33 Ser/Thr phosphorylation sites in 15 subunits of the proteasome. The data obtained by us and other groups reveal that the 26S proteasome contains at least 88 phospho‐amino acids including 63 pSer, 23 pThr, and 2 pTyr residues. Dephosphorylation treatment of the 19S RP with λ phosphatase resulted in a 30% decrease in ATPase activity, demonstrating that phosphorylation is involved in the regulation of ATPase activity in the proteasome. Third, we tried to detect glycosylated subunits of the 26S proteasome. However, we identified neither N‐ and O‐linked oligosaccharides nor O‐linked β‐N‐acetylglucosamine in the 19S RP and 20S proteasome subunits. To date, a total of 110 co‐ and post‐translational modifications, including Nα‐acetylation, Nα‐myristoylation, and phosphorylation, in the yeast 26S proteasome have been identified.

Isoelectric focusing of high-molecular-weight protein complex under native conditions using agarose gel

The isolation and characterization of protein complexes are essential steps toward understanding cellular functions. A method for separating and characterizing high-molecular-weight protein complexes using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) with native agarose gel isoelectric focusing (IEF) is described. Using this method, fractions containing high-molecular-weight protein complexes were analyzed. The advantages of using native agarose gel IEF include the ability to concentrate the protein complexes and the ease of handling when performing 2D separations. Although limited with respect to the size of molecules and particles that may be separated, this method is useful for the isolation and characterization of high-molecular-weight protein complexes.

Multiplex detection and identification of proteins on a PVDF membrane blocked with a synthetic polymer-based reagent

2‐DE is one of the most powerful methods for analyzing proteins expressed in cells and tissues. Immunodetection of proteins blotted on a polymer membrane is the method of choice for detecting specific proteins in 2‐D gels. To precisely locate spots of immunoreactive proteins in 2‐D gels, both dye staining and immunodetection were performed on the same PVDF membrane. Prior to immunodetection, nonspecific adsorption of the antibodies to the membrane was blocked with a synthetic polymer‐based reagent (N‐102) after protein transfer. The protein was then stained with colloidal gold or CBB followed by protein spot identification by LC‐MS. Described herein is a method for multiplex analysis of proteins transferred to a PVDF membrane. Proteins that were phosphorylated at tyrosine in the phosphoproteome of rice callus or human ovarian cancer cells were detected by immunoblotting and subsequently identified with high precision.