Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

The taxonomic position of two butane-utilizing bacteria was studied using a polyphasic approach. Biochemical and physiological characteristics indicated these to be members of the genus Pseudomonas, showing more similarity to Pseudomonas mendocina than to any other species. The major fatty acids found in these two strains also pointed to their similarity to P. mendocina. On the other hand, DNA-DNA hybridization studies with seven related Pseudomonas species belonging to the gamma-Proteobacteria and the deltaTm values of reassociated molecules clearly showed that these two strains do not belong to any of the seven species tested. The 16S rRNA gene was sequenced and compared with the sequences available in the GenBank database. Phylogenetic analysis using the region covering positions 31-1488 (Escherichia coli numbering) confirmed these observations and placed these two strains as members of the authentic Pseudomonas, but not in any existing species of the genus. On the basis of biochemical characteristics, fatty acid profiles, DNA-DNA reassociation and deltaTm values, as well as 16S rRNA gene sequence analyses, these two isolates were shown to belong to one species but to have characteristics distinct from those of validly described species of Pseudomonas (sensu stricto). These strains, therefore, should be recognized as a novel species, for which the name Pseudomonas indica sp. nov. is proposed. The type strain is strain IMT37T (= MTCC 3713T = DSM 14015T).

Phospholipid scramblases externalize phosphatidylserine to facilitate numerous physiological processes. Several members of the structurally unrelated TMEM16 and G protein-coupled receptor (GPCR) protein families mediate phospholipid scrambling. The structure of a TMEM16 scramblase shows a membrane-exposed hydrophilic cavity, suggesting that scrambling occurs via the ‟credit-card" mechanism where lipid headgroups permeate through the cavity while their tails remain associated with the membrane core. Here we show that afTMEM16 and opsin, representatives of the TMEM16 and GCPR scramblase families, transport phospholipids with polyethylene glycol headgroups whose globular dimensions are much larger than the width of the cavity. This suggests that transport of these large headgroups occurs outside rather than within the cavity. These large lipids are scrambled at rates comparable to those of normal phospholipids and their presence in the reconstituted vesicles promotes scrambling of normal phospholipids. This suggests that both large and small phospholipids can move outside the cavity. We propose that the conformational rearrangements underlying TMEM16- and GPCR-mediated credit-card scrambling locally deform the membrane to allow transbilayer lipid translocation outside the cavity and that both mechanisms underlie transport of normal phospholipids.

Several class-A G protein-coupled receptor (GPCR) proteins act as constitutive phospholipid scramblases catalyzing the transbilayer translocation of >10,000 phospholipids per second when reconstituted into synthetic vesicles. To address the molecular mechanism by which these proteins facilitate rapid lipid scrambling, we carried out large-scale ensemble atomistic molecular dynamics simulations of the opsin GPCR. We report that, in the process of scrambling, lipid head groups traverse a dynamically revealed hydrophilic pathway in the region between transmembrane helices 6 and 7 of the protein while their hydrophobic tails remain in the bilayer environment. We present quantitative kinetic models of the translocation process based on Markov State Model analysis. As key residues on the lipid translocation pathway are conserved within the class-A GPCR family, our results illuminate unique aspects of GPCR structure and dynamics while providing a rigorous basis for the design of variants of these proteins with defined scramblase activity.

The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein-related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1-Lam6, in the yeast Lam1-Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling "head-first" entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress.