Conservation of gene function in the solanaceae as revealed by comparative mapping of domestication traits in eggplant

Quantitative trait loci (QTL) for domestication-related traits were identified in an interspecific F(2) population of eggplant (Solanum linnaeanum x S. melongena). Although 62 quantitative trait loci (QTL) were identified in two locations, most of the dramatic phenotypic differences in fruit weight, shape, color, and plant prickliness that distinguish cultivated eggplant from its wild relative could be attributed to six loci with major effects. Comparison of the genomic locations of the eggplant fruit weight, fruit shape, and color QTL with the positions of similar loci in tomato, potato, and pepper revealed that 40% of the different loci have putative orthologous counterparts in at least one of these other crop species. Overall, the results suggest that domestication of the Solanaceae has been driven by mutations in a very limited number of target loci with major phenotypic effects, that selection pressures were exerted on the same loci despite the crops' independent domestications on different continents, and that the morphological diversity of these four crops can be explained by divergent mutations at these loci.     

Stabilization of the biotinoyl domain of Escherichia coli acetyl-CoA carboxylase by interactions between the attached biotin and the protruding "thumb" structure

We previously reported (Chapman-Smith, A., Forbes, B. E., Wallace, J. C., and Cronan, J. E., Jr. (1997) J. Biol. Chem. 272, 26017-26022) that the biotinylated (holo) species of the biotin carboxyl carrier protein (BCCP) biotinoyl domain is much more resistant to chemical modification and proteolysis than the unbiotinylated (apo) form. We hypothesized that the increased stability was due to a conformational change engendered by interaction of the domain with biotin protein ligase, the enzyme that attaches the biotin moiety. We now report that a BCCP-87 species to which the biotin moiety was attached by chemical acylation rather than by biotin protein ligase showed the characteristically greater stability of the holo biotinoyl domain. This result demonstrates that our hypothesis was incorrect; the attached biotin is solely responsible for the increased stability. The bacterial and chloroplast multisubunit acetyl-CoA carboxylases are unusual in that the highly symmetrical and conserved structure of the biotinoyl domain of the BCCP subunit is disrupted by a structured loop called the "thumb" that protrudes from body of the domain. Prior structural work showed that the thumb interacts with uriedo ring of the attached biotin moiety. We have tested whether the thumb-biotin interactions are responsible for the greater holo form stability by examination of two BCCP-87 species that lack the thumb. These BCCP species were produced in both the apo and holo forms, and their sensitivities to trypsin digestion were compared. The holo forms of these proteins were found to be only marginally more stable than their apo forms and much more sensitive to trypsin digestion than the wild type holo-BCCP-87. Therefore, removal of the thumb has an effect similar to lack of biotinylation, indicating that thumb-biotin interactions are responsible for most (but not all) of the increased stability of the holo biotinoyl domain. In the course of these experiments we demonstrated that treatment of Escherichia coli with the peptide deformylase inhibitor, actinonin, results in the expected (but previously unreported) accumulation of an N-formylated protein species.     

The RAVE complex is essential for stable assembly of the yeast V-ATPase

Vacuolar proton-translocating ATPases are composed of a peripheral complex, V(1), attached to an integral membrane complex, V(o). Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384-391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V(1) complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V(1) sectors in a mutant lacking V(o) sectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE small middle dotV(1) interactions are specifically disrupted in cells lacking V(1) subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE small middle dotV(1) binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly.     

Inducible nitric-oxide synthase attenuates vasopressin-dependent Ca2+ signaling in rat hepatocytes

Increases in both Ca(2+) and nitric oxide levels are vital for a variety of cellular processes; however, the interaction between these two crucial messengers is not fully understood. Here, we demonstrate that expression of inducible nitric-oxide synthase in hepatocytes, in response to inflammatory mediators, dramatically attenuates Ca(2+) signaling by the inositol 1,4,5-trisphosphate-forming hormone, vasopressin. The inhibitory effects of induction were reversed by nitric oxide inhibitors and mimicked by prolonged cyclic GMP elevation. Induction was without effect on Ca(2+) signals in response to AlF(4)(-) or inositol 1,4,5-trisphosphate, indicating that phospholipase C activation and release of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive Ca(2+) stores were not targets for nitric oxide inhibition. Vasopressin receptor levels, however, were dramatically reduced in induced cultures. Our data provide a possible mechanism for hepatocyte dysfunction during chronic inflammation.     

Nuclear injection of anti-pigpen antibodies inhibits endothelial cell division

Endothelial cell proliferation is required for angiogenesis in both embryonic and adult tissues. In rat brain tumors, it has recently been shown that the nuclear protein pigpen is expressed selectively in endothelial cells of developing microvasculature but not in the established peritumoral vessels (Blank, M., Weinschenk, T., Priemer, M., and Schluesener, H. (2001) J. Biol. Chem. 276, 16464-16468). This finding suggests that pigpen may be important for promoting the undifferentiated, or "angiogenic" endothelial cell phenotype. Our studies show that pigpen protein and mRNA are expressed in actively dividing endothelial cells and down-regulated as they become confluent. Protein distribution is regulated in a cell cycle-dependent manner. We conclude that this expression pattern is important for and not simply ancillary to proliferation because nuclear microinjection of anti-pigpen Fab fragments inhibited endothelial cell division. Moreover, expression of the proliferating cell marker Ki67 was inhibited in antibody-injected cells. The absence of Ki67 suggests exit from rather than arrest within (for example, at the G(1)/S interface) the cell cycle. Together with earlier observations on the structure and expression of this molecule, our data support the hypothesis that pigpen helps regulate endothelial cell differentiation state.     

Lipid free apolipoprotein E binds to the class B Type I scavenger receptor I (SR-BI) and enhances cholesteryl ester uptake from lipoproteins

The Class B type I scavenger receptor I (SR-BI) is a physiologically relevant high density lipoprotein (HDL) receptor that can mediate selective cholesteryl ester (CE) uptake by cells. Direct interaction of apolipoprotein E (apoE) with this receptor has never been demonstrated, and its implication in CE uptake is still controversial. By using a human adrenal cell line (NCI-H295R), we have addressed the role of apoE in binding to SR-BI and in selective CE uptake from lipoproteins to cells. This cell line does not secrete apoE and SR-BI is its major HDL-binding protein. We can now provide evidence that 1) free apoE is a ligand for SR-BI, 2) apoE associated to lipids or in lipoproteins does not modulate binding or CE-selective uptake by the SR-BI pathway, and 3) the direct interaction of free apoE to SR-BI leads to an increase in CE uptake from lipoproteins of both low and high densities. We propose that this direct interaction could modify SR-BI structure in cell membranes and potentiate CE uptake.     

Myosin VIIa,harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.     

Caspase-10 is recruited to and activated at the native TRAIL and CD95 death-inducing signalling complexes in a FADD-dependent manner but can not functionally substitute caspase-8

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.