Hideyoshi Yokosawa

Production and characterization of monoclonal antibodies specific to multi-ubiquitin chains of polyubiquitinated proteins

Polyubiquitinated proteins tagged with multi‐ubiquitin chains are substrates preferred by the 26 S proteasome (a ubiquitin/ATP‐dependent proteolytic complex). Here, we developed a simple method for the efficient preparation of polyubiquitinated proteins which are degraded by the 26 S proteasome in an ATP‐dependent manner. Our efficient method enabled us to produce ten monoclonal antibodies that recognized the multi‐ubiquitin chains of the polyubiquitinated proteins, but not free ubiquitin or the protein moieties. Eight of the antibodies recognized only the multi‐ubiquitin chains of the polyubiquitinated proteins, while the other two antibodies cross‐reacted with mono‐ubiquitin and methyl‐ubiquitin, both of which are linked to proteins via an isopeptide bond, as well as with the multi‐ubiquitin chains. Thus these antibodies are novel and useful tools for the identification and quantification of polyubiquitinated proteins in various cells and tissues under physiological and pathological conditions.

Cell cycle-dependent change of proteasome distribution during embryonic development of the ascidian Halocynthia roretzi

The proteasome is a multicatalytic proteinase complex composed of nonidentical subunits. By immunocytochemical analysis using monoclonal antibody raised against the egg proteasome, we demonstrate that the proteasome undergoes changes in its subcellular distribution, depending on the cell division cycle during embryonic development of the ascidian Halocynthia roretzi. During interphase, the proteasome is localized in the nucleus, i.e., in the nucleoplasm and along the nuclear membrane. The proteasome disappears from the nucleoplasm in prophase and from the nuclear envelope in prometaphase. During early metaphase, the proteasome is detectable in the chromosomes and, at late stages of metaphase, the immunoreactivity also occurs in the peripheral region of each spindle pole and at the mitotic spindle. In anaphase, however, the staining disappears in the mitotic apparatus. In telophase, the proteasome is again localized in the newly formed nucleus. In addition to the localization in the nucleus and around the mitotic apparatus, the proteasome shows cytoplasmic localization throughout the cell division cycle. Such a change of subcellular distribution of the proteasome is clearly demonstrated in the synchronously dividing blastomeres and also is believed to occur in the postcleavage embryos. These observations suggest that the proteasome may play a key role in the progression of cell division cycle.

Different ratios in 20 S proteasomes and regulatory subunit complexes in two isoforms of the 26 S proteasome purified from rabbit skeletal muscle

A ubiquitin/ATP‐dependent proteinase complex (26 S proteasome) was highly purified from rabbit skeletal muscle. The purified 26 S proteasome easily dissociated into a 20 S proteasome and a regulatory subunit complex on non‐denaturing PAGE. By using cleavable and non‐cleavable cross‐linkers, it was revealed that the 26 S proteasome exists in two isoforms: one (D complex) consists of the 20 S proteasome and the regulatory subunit complex in the ratio of one to two, while the other (C complex) exists in an equal molar ratio. Molecular masses of the former and the latter isoforms were estimated to be 1,700 kDa and 1,400 kDa, respectively, by gel filtration, and 2,400 kDa and 1,400 kDa, respectively, by Ferguson plot analysis. Furthermore, both isoforms efficiently hydrolyzed Suc‐Leu‐Leu‐Val‐Tyr‐MCA and ubiquitin‐conjugated [125I]lysozyme. These results suggest that the D and C complexes are active proteinase complexes, most probably corresponding to the dumbbell‐like and mushroom‐like (or space capsule‐like) molecules, respectively.

The 26 S proteasome is activated at two points in the ascidian cell cycle.

The proteasome undergoes cell cycle‐dependent changes in its subcellular distribution during ascidian embryonic development [(1992) Dev. Biol. 151, 27–33]. In this study, we demonstrate that the 26 S proteasome is markedly activated in both prophase and metaphase of the mitotic cell cycle in the ascidian embryos in comparison with the case of the 20 S proteasome. These results suggest that the 26 S proteasome is activated and participates in proteolysis at the restricted stages of the cell cycle.

Comparative studies on proteasomes (multicatalytic proteinases) isolated from spermatozoa and eggs of sea urchins.

1. Proteasomes (multicatalytic proteinases) have been purified from spermatozoa and eggs of sea urchins by successive chromatographies on DEAE-cellulose, hydroxylapatite, and Sepharose 6B. 2. The isolated sperm and egg proteasomes showed similar properties such as molecular weight (both 630,000) and multiple catalytic activity (pH optimum, substrate specificity, inhibitor-susceptibility and SDS-stimulation). 3. Distinct protein components were detected between sperm and egg proteasomes by SDS-polyacrylamide gel electrophoresis and immunoblot analysis using antibody against egg proteasome.