The proteasome subunit, C2, contains an important site for binding of the PA28 (11S) activator.

The PA28, or 11S regulatory complex, stimulates the peptidase activities of the 20S proteasome. Monoclonal antibodies were screened for their ability to inhibit the activation by PA28 of proteasomes from rabbit reticulocytes. We identified one antibody that inhibited proteasome activation by PA28 and dissociated formed proteasome-PA28 complexes. A fourfold molar excess of antibody to proteasome markedly reduced the PA28 activation of three peptidase activities. Examination of proteasome-antibody mixtures by electron microscopy revealed that the antibody formed chains of proteasomes, and digital image analysis of individual proteasomes demonstrated that the antibody binds to the outer alpha rings. This antibody recognizes proteasome subunit C2, which we conclude contains an important contact site for the PA28 activator. However, the antibody did not block proteasome activation by PA700, or 19S regulator, which also associates with the alpha rings. Thus, these two regulators appear to bind to the proteasome at different sites.

Degradation of monoubiquitinated alpha-globin by 26S proteasomes.

Ubiquitin-125I-alpha-globin conjugate fractions containing either one (Ub1-alpha), or two (Ub2-alpha), or a mixture of three and four (Ub3,4-alpha) molecules of ubiquitin (Ub), covalently linked to one 125I-alpha-globin molecule were isolated after incubation of a proteolysis reaction mixture containing ATP, ubiquitin aldehyde-treated reticulocyte lysate, and human 125I-alpha-globin. Each of the purified conjugate fractions or an identically-purified control sample of unconjugated 125I-alpha-globin was incubated as a substrate in companion proteolysis reaction mixtures containing either purified 26S or 20S rabbit reticulocyte proteasomes. The initial rate of ATP-dependent degradation of the Ub1-alpha conjugate by the 26S proteasomes was approximately 0.44% (1.1 fmol)/min while that of the free 125I-alpha-globin was undetectable. The initial rates of ATP-dependent degradation by the 26S proteasomes of the Ub2-alpha and Ub3,4-alpha conjugates were 2- to-3-fold that of the Ub1-alpha species. Conversely, the degradation of free 125I-alpha-globin and its ubiquitinated conjugates by the 20S proteasomes was not dependent on ATP, nor did it increase with the size of the Ub adduct. Analysis of the products of a reaction mixture with 26S proteasomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed no conversion of the Ub1-alpha conjugate substrate to higher-molecular-mass conjugates. These results suggest that monobiquitinated alpha-globin can be degraded significantly and specifically by interaction directly with the 26S proteasomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and function of the Drosophila gene runt in early stages of neural development.

The Drosophila gene runt was initially identified on the basis of its role during segmentation. Recent molecular and genetic studies have demonstrated that the runt gene encodes a novel nuclear protein whose developmental importance is not exclusive to segmentation. This report addresses the functional relevance of runt expression in the developmental pathway of neurogenesis. Antibodies against the runt protein reveal that it is expressed in a subset of neuroblasts, ganglion-mother cells and neurons. A subset of these neurons also co-express the segmentation gene even-skipped (eve). Using eve as a marker, we show that runt is required for the normal development of these neurons. A runt P-transposon that lacks neural cis-regulatory elements is used to show that these neurons require runt activity independent of its activity during segmentation. These results are confirmed using a temperature-sensitive runt allele. Further temperature-shift experiments indicate that the requirement for runt is during an early stage of neurogenesis. Based on its pattern of expression and its temporal requirements, runt is distinguished as one of the earliest acting genes involved in the generation of diverse cell fates in the developing Drosophila nervous system.

The Drosophila segmentation gene runt encodes a novel nuclear regulatory protein that is also expressed in the developing nervous system.

Generation of the anterior-posterior body pattern in the Drosophila embryo requires the activity of the segmentation genes. The segmentation gene runt has been classified as one of the primary pair-rule genes because of the pivotal role it plays in regulating the expression of other pair-rule genes. Here, we present the structure of this gene and describe the pattern of runt protein expression during embryogenesis. The deduced protein sequence shows no obvious overall homology with any sequences in the data base. The absence of an identifiable transcription factor motif (e.g., homeo box, zinc finger, leucine zipper, or helix-loop-helix) makes runt different from the other early-acting segmentation proteins. A runt-specific polyclonal antibody was generated and used to demonstrate that the subcellular location of the protein is in the nucleus. Double-staining immunolocalization experiments were used to determine the overlap of the runt protein pattern with the patterns of the pair-rule genes hairy (h), even-skipped (eve), and fushi tarazu (ftz). We found that the patterns of runt and hairy are complementary. Their phasing is shifted anteriorly by two cell diameters with respect to the complementary eve and ftz patterns. Experiments with the runt antibody also indicated that the protein is present throughout embryogenesis and is expressed extensively in the developing central and peripheral nervous system.