Multiple gains of spliceosomal introns in a superfamily of vertebrate protease inhibitor genes

Background  

Intron gains reportedly are very rare during evolution of vertebrates, and the mechanisms underlying their creation are largely unknown. Previous investigations have shown that, during metazoan radiation, the exon-intron patterns of serpin superfamily genes were subject to massive changes, in contrast to many other genes.     

Jumping mode atomic force microscopy on grana membranes from spinach

The thylakoid membrane system is a complex membrane system that organizes and reorganizes itself to provide plants optimal chemical energy from sunlight under different and varying environmental conditions. Grana membranes are part of this system and contain the light-driven water-splitting enzyme Photosystem II (PSII) and light-harvesting antenna complexes. Here, we present a direct visualization of PSII complexes within grana membranes from spinach. By means of jumping mode atomic force microscopy in liquid, minimal forces were applied between the scanning tip and membrane or protein, allowing complexes to be imaged with high detail. We observed four different packing arrangements of PSII complexes, which occur primarily as dimers: co-linear crystalline rows, nanometric domains of straight or skewed rows, and disordered domains. Upon storing surface-adhered membranes at low temperature prior to imaging, large-scale reorganizations of supercomplexes between PSII and light-harvesting complex II could be induced. The highest resolution images show the existence of membrane domains without obvious topography extending beyond supercomplexes. These observations illustrate the possibility for diffusion of proteins and smaller molecules within these densely packed membranes.     

Oncogenic function for the Dlg1 mammalian homolog of the Drosophila discs-large tumor suppressor

The fact that several different human virus oncoproteins, including adenovirus type 9 E4-ORF1, evolved to target the Dlg1 mammalian homolog of the membrane-associated Drosophila discs-large tumor suppressor has implicated this cellular factor in human cancer. Despite a general belief that such interactions function solely to inactivate this suspected human tumor suppressor protein, we demonstrate here that E4-ORF1 specifically requires endogenous Dlg1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. Based on our results, we propose a model wherein E4-ORF1 binding to Dlg1 triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation. These findings establish the first known function for Dlg1 in virus-mediated cellular transformation and also surprisingly expose a previously unrecognized oncogenic activity encoded by this suspected cellular tumor suppressor gene.     

Protein shape and crowding drive domain formation and curvature in biological membranes

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization are known under physiological conditions are of great value. We therefore conducted atomic force microscopy and polarized spectroscopy experiments on membranes of the photosynthetic bacterium Rhodobacter sphaeroides. These membranes are densely populated with peripheral light harvesting (LH2) complexes, physically and functionally connected to dimeric reaction center-light harvesting (RC-LH1-PufX) complexes. Here, we show that even when converting the dimeric RC-LH1-PufX complex into RC-LH1 monomers by deleting the gene encoding PufX, both the appearance of protein domains and the associated membrane curvature are retained. This suggests that a general mechanism may govern membrane organization and shape. Monte Carlo simulations of a membrane model accounting for crowding and protein geometry alone confirm that these features are sufficient to induce domain formation and membrane curvature. Our results suggest that coexisting ordered and fluid domains of like proteins can arise solely from asymmetries in protein size and shape, without the need to invoke specific interactions. Functionally, coexisting domains of different fluidity are of enormous importance to allow for diffusive processes to occur in crowded conditions.     

The organization of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus and the structural role of carotenoids and protein

The organization of bacteriochlorophyll c (BChl c) molecules was studied in normal and carotenoid-deficient chlorosomes isolated from the green phototrophic bacterium Chloroflexus aurantiacus. Carotenoid-deficient chlorosomes were obtained from cells grown in the presence of 60 µg of 2-hydroxybiphenyl per ml. At this concentration, BChl c synthesis was not affected while the formation of the 5.7 kDa chlorosome polypeptide was inhibited by about 50% (M. Foidl et al., submitted). Absorption, linear dichroism and circular dichroism spectroscopy showed that the organization of BChl c molecules with respect to each other as well as to the long axis of the chlorosomes was similar for both types of chlorosomes. Therefore, it is concluded that the organization of BChl c molecules is largely independent on the presence of the bulk of carotenoids as well as of at least half of the normal amount of the 5.7 kDa polypeptide. The Stark spectra of the chlorosomes, as characterized by a large difference polarizability for the ground- and excited states of the interacting BChl c molecules, were much more intense than those of individual pigments. It is proposed that this is caused by the strong overlap of BChl c molecules in the chlorosomes. In contrast to individual chlorophylls, BChl c in chlorosomes did not give rise to a significant difference permanent dipole moment for the ground- and excited states. This observation favors models for the BChl c organization which invoke the anti-parallel stacking of linear BChl c aggregates above those models in which linear BChl c aggregates are stacked in a parallel fashion. The difference between the Stark spectrum of carotenoid-deficient and WT chlorosomes indicates that the carotenoids are in the vicinity of the BChls.     

Flexibility and size heterogeneity of the LH1 light harvesting complex revealed by atomic force microscopy: functional significance for bacterial photosynthesis

Previous electron microscopic studies of bacterial RCLH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q(B) site of the RC to the quinone pool prior to reduction of the cytochrome bc(1) complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (alpha(1)beta(1)Bchl(2)) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process.     

Atypical dissemination of the highly neurotropic Borna disease virus during persistent infection in cyclosporine A-treated, immunosuppressed rats

In adult rats infected with Borna disease virus, the virus was found exclusively in the brain, whereas in cyclosporine A-treated rats, infectious virus was also detected in peripheral nerve fibers and, unexpectedly, in adjacent organ-specific cells. In contrast to untreated virus-infected rats, no major histocompatibility complex class II expression was found in the brain of cyclosporine A-treated animals.