Occurrence of a putative SCF ubiquitin ligase complex in Drosophila.

Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. We have identified in Drosophila melanogaster the closest homologues to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5. We show that putative Drosophila SCF core subunits dSkpA and dRbx1 both interact directly with dCu11 and the F-box protein Slmb. We also describe the direct interaction of the UbcH5 related protein UbcD1 with dCul1 and Slmb. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant showed that Drosophila Rbx1 is able to restore the yeast cells viability. Our results suggest that dRbx1, dSkpA, dCullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1.

Inhibition of UDP-glucose: protein transglucosylase by a maize endosperm protein factor.

UDP-glucose: protein transglucosylase (UPTG, EC 2.4.1.112) catalyzes the first step of protein-bound alpha-glucan synthesis in potato tuber and developing maize endosperm. The presence of a non-dialyzable, heat labile protein responsible for low levels of UPTG activity in developing maize endosperm was investigated. UPTG activity in 5-day old maize seedlings and potato tuber solubilized preparations was also reduced by the endosperm preparation. FPLC-Mono Q column chromatography of developing maize endosperm was effective in separating the inhibitor protein (IP) from UPTG. After gel filtration on Superose 12, IP yielded a major polypeptide of about 80 kDa on SDS-PAGE. IP was purified by gel filtration on Superose 12 and preparative SDS-PAGE, and specific antibodies were prepared. Polyclonal antibodies reacted specifically with an 80 kDa polypeptide of developing maize endosperm on Western blot. They also recognized a similar band in 5-day old maize seedlings, but not in potato tubers. The identification of a factor that regulates the level of UPTG activity in developing maize endosperm may help to elucidate the functional role of the enzyme in the initiation of starch synthesis during seed development.

Sequence-specific DNA modification in Acetobacter xylinum.

Two cryptic plasmids have been discovered in Acetobacter xylinum B42 and in its derivative PEA-1, a cellulose defective mutant. These two plasmids were designated pAX1 and pAX2 (50 and 105 kb in size, respectively). A restriction map was constructed for pAX1. Attempts to cure these plasmids were unsuccessful. Enzyme restriction analysis showed that these plasmids contain protected EcoRI and ApoI sites. Using Southern blot and hybridization techniques, the protection was extended to chromosomal DNA. Enzyme restriction analysis of several plasmids, from different origins and containing different incompatibility groups, isolated from strain PEA-1 also showed EcoRI and ApoI protection. The presence of modifications on specific sequences was not found in A. xylinum 8747. These results strongly suggest the presence of a modification system in A. xylinum B42 that recognizes the tetranucleotide 5'-AATT.