Phospholipid transport: sighting a new face of an old friend

A recent study now shows that opsin catalyzes rapid movement of phospholipids from one leaflet of a membrane bilayer to the other. This capability illuminates a mechanism for this physiologically important process.     

Systematic chromosomal deletion of bacterial ribosomal protein genes

Detailed studies of ribosomal proteins (RPs), essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual RP genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, we found nine RPs (L15, L21, L24, L27, L29, L30, L34, S9, and S17) nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli RP genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function and may ultimately allow systematic substitution of RPs with RNA.     

A simple cell based assay to measure Parkin activity

Parkin is an ubiquitin-protein ligase mutated in Autosomal Recessive - Juvenile Parkinsonism. Here, we describe a cell-based assay to measure Parkin's ubiquitin-protein ligase activity. It relies on the ability of Parkin to recognise depolarised mitochondria and exploits a cell line where Parkin expression is inducible. In these cells, Parkin expression promotes mitophagy and accelerates cell death in response to mitochondrial depolarisers. Time-lapse imaging confirmed cell death and revealed increased perinuclear mitochondrial clustering following induction of Parkin expression in cells exposed to carbonyl cyanide m-chlorophenylhydrazone. Similar effects were not observed with α-synuclein or DJ-1, other proteins associated with the development of Parkinson's disease, confirming the specificity of the assay. We have used this assay to demonstrate that ligase-defective Parkin mutants are inactive, and cellular proteasomal activity (using the proteasomal inhibitors MG132, clasto-lactacystin β-lactone and epoxomicin) is essential for the Parkin mediated effect. As the assay is suitable for high-throughput screening, it has the potential to identify novel proteostasis compounds that stimulate the activity of Parkin mutants for therapeutic purposes, to identify modulators of kinase activities that impact on Parkin function, and to act as a functional read-out in reverse genetics screens aimed at identifying modifiers of Parkin function during mitophagy.     

Genetic confirmation of a hybrid between two highly divergent cardinalid species: A rose‐breasted grosbeak (Pheucticus ludovicianus) and a scarlet tanager (Piranga olivacea)

Using low‐coverage whole‐genome sequencing, analysis of vocalizations, and inferences from natural history, we document a first‐generation hybrid between a rose‐breasted grosbeak (Pheucticus ludovicianus) and a scarlet tanager (Piranga olivacea). These two species occur sympatrically throughout much of eastern North America, although were not previously known to interbreed. Following the field identification of a putative hybrid, we use genetic and bioacoustic data to show that a rose‐breasted grosbeak was the maternal parent and a scarlet tanager was the paternal parent of the hybrid, whose song was similar to the latter species. These two species diverged >10 million years ago, and thus it is surprising to find a hybrid formed under natural conditions in the wild. Notably, the hybrid has an exceptionally heterozygous genome, with a conservative estimate of a heterozygous base every 100 bp. The observation that this hybrid of such highly divergent parental taxa has survived until adulthood serves as another example of the capacity for hybrid birds to survive with an exceptionally divergent genomic composition.