Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.

Cold Microwave-Enabled Protein Detection and Quantification.

Protein screening/detection is an essential tool in many laboratories. Owing to the relatively large time investments that are required by standard protocols, the development of methods with higher throughput while maintaining an at least comparable signal-to-noise ratio is highly beneficial in many research areas. This chapter describes how cold microwave technology can be used to enhance the rate of molecular interactions and provides protocols for dot blots, Western blots, and ELISA procedures permitting a completion of all incubation steps (blocking and antibody steps) within 24-45 min.

Evidence for direct squalene and 2,3-oxidosqualene binding by supernatant protein factor

We present the crystal structures of the SEC14-like domain of supernatant protein factor (SPF) in complex with squalene and 2,3-oxidosqualene. The structures were resolved at 1.75 Å (complex with squalene) and 1.6 Å resolution (complex with 2,3-oxidosqualene), leading in both cases to clear images of the protein/ substrate interactions. Ligand binding is facilitated by removal of the Golgi-dynamics (GOLD) C-terminal domain of SPF, which, as shown in previous structures of the apo-protein, blocked the opening of the binding pocket to the exterior. Both substrates bind into a large hydrophobic cavity, typical of such lipid-transporter family. Our structures report no specific recognition mode for the epoxide group. In fact, for both molecules, ligand affinity is dominated by hydrophobic interactions, and independent investigations by computational models or differential scanning micro-calorimetry reveal similar binding affinities for both ligands. Our findings elucidate the molecular bases of the role of SPF in sterol endo-synthesis, supporting the original hypothesis that SPF is a facilitator of substrate flow within the sterol synthetic pathway. Moreover, our results suggest that the GOLD domain acts as a regulator, as its conformational displacement must occur to favor ligand binding and release during the different synthetic steps.

Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process

Owing to their pathogenical role and unique ability to exist both as soluble proteins and transmembrane complexes, pore-forming toxins (PFTs) have been a focus of microbiologists and structural biologists for decades. PFTs are generally secreted as water-soluble monomers and subsequently bind the membrane of target cells. Then, they assemble into circular oligomers, which undergo conformational changes that allow membrane insertion leading to pore formation and potentially cell death. Aerolysin, produced by the human pathogen Aeromonas hydrophila, is the founding member of a major PFT family found throughout all kingdoms of life. We report cryo-electron microscopy structures of three conformational intermediates and of the final aerolysin pore, jointly providing insight into the conformational changes that allow pore formation. Moreover, the structures reveal a protein fold consisting of two concentric β-barrels, tightly kept together by hydrophobic interactions. This fold suggests a basis for the prion-like ultrastability of aerolysin pore and its stoichiometry.