The Versatile Molecular Complex Component LC8 Promotes Several Distinct Steps of Flagellar Assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3’s LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly

Microbial engineering for production of N-functionalized amino acids and amines

Mindt M, Walter T, Kugler P, Wendisch VF. Microbial engineering for production of N-functionalized amino acids and amines. Biotechnology Journal . 2020;15(7): 1900451.N‐ functionalized amines play important roles in nature and occur, for example, in the antibiotic vancomycin, the immunosuppressant cyclosporine, the cytostatic actinomycin, the siderophore aerobactin, the cyanogenic glucoside linamarin, and the polyamine spermidine. In the pharmaceutical and fine‐chemical industries N‐ functionalized amines are used as building blocks for the preparation of bioactive molecules. Processes based on fermentation and on enzyme catalysis have been developed to provide sustainable manufacturing routes to N‐ alkylated, N‐ hydroxylated, N‐ acylated, or other N‐ functionalized amines including polyamines. Metabolic engineering for provision of precursor metabolites is combined with heterologous N‐ functionalizing enzymes such as imine or ketimine reductases, opine or amino acid dehydrogenases, N‐ hydroxylases, N‐ acyltransferase, or polyamine synthetases. Recent progress and applications of fermentative processes using metabolically engineered bacteria and yeasts along with the employed enzymes are reviewed and the perspectives on developing new fermentative processes based on insight from enzyme catalysis are discussed

MutL traps MutS at a DNA mismatch

DNA mismatch repair is the process by which errors generated during DNA replication are corrected. Mutations in the proteins that initiate mismatch repair, MutS and MutL, are associated with greater than 80% of hereditary nonpolyposis colorectal cancer (HNPCC) and many sporadic cancers. The assembly of MutS and MutL at a mismatch is an essential step for initiating repair; however, the nature of these interactions is poorly understood. Here, we have discovered that MutL fundamentally changes the properties of mismatch-bound MutS by preventing it from sliding away from the mismatch, which it normally does when isolated. This finding suggests a mechanism for localizing the activity of repair proteins near the mismatch

Proteomic analysis of the Plasmodium male gamete reveals the key role for glycolysis in flagellar motility.

BACKGROUND: Gametogenesis and fertilization play crucial roles in malaria transmission. While male gametes are thought to be amongst the simplest eukaryotic cells and are proven targets of transmission blocking immunity, little is known about their molecular organization. For example, the pathway of energy metabolism that power motility, a feature that facilitates gamete encounter and fertilization, is unknown. METHODS: Plasmodium berghei microgametes were purified and analysed by whole-cell proteomic analysis for the first time. Data are available via ProteomeXchange with identifier PXD001163. RESULTS: 615 proteins were recovered, they included all male gamete proteins described thus far. Amongst them were the 11 enzymes of the glycolytic pathway. The hexose transporter was localized to the gamete plasma membrane and it was shown that microgamete motility can be suppressed effectively by inhibitors of this transporter and of the glycolytic pathway. CONCLUSIONS: This study describes the first whole-cell proteomic analysis of the malaria male gamete. It identifies glycolysis as the likely exclusive source of energy for flagellar beat, and provides new insights in original features of Plasmodium flagellar organization

Clamp loader ATPases and the evolution of DNA replication machinery

Clamp loaders are pentameric ATPases of the AAA+ family that operate to ensure processive DNA replication. They do so by loading onto DNA the ring-shaped sliding clamps that tether the polymerase to the DNA. Structural and biochemical analysis of clamp loaders has shown how, despite differences in composition across different branches of life, all clamp loaders undergo the same concerted conformational transformations, which generate a binding surface for the open clamp and an internal spiral chamber into which the DNA at the replication fork can slide, triggering ATP hydrolysis, release of the clamp loader, and closure of the clamp round the DNA. We review here the current understanding of the clamp loader mechanism and discuss the implications of the differences between clamp loaders from the different branches of life

MutL traps MutS at a DNA mismatch

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (β-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL’s endonuclease activity by β-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS–MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with β-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability