Interaction of a coupling factor from Rhodospirillum rubrum with coupling factor deficient chromatophores.

A coupling factor necessary for the photophosphorylation and Mg2+-ATPase activities in Rhodospirillum rubrum chromatophores has been separated from these particles. Although the redox potential of coupling factor deficient chromatophores is slightly more oxidized than of the control, the addition of the coupling factor for reconstitution does not alter the redox potential. Phenazine methosulfate cannot restore or significantly enhance the photophosphorylation activities of uncoupled or reconstituted chromatophores compared to the control. The coupling factor can bind to coupling factor deficient membranes without addition of magnesium ions and thus restore the photophosphorylation and Mg2+-ATPase activities of these vesicles. The Ca2+-ATPase in the coupling factor preparation shows binding characteristics similar to those of the coupling factor.     

Low codon bias and high rates of synonymous substitution in Drosophila hydei and D. melanogaster histone genes

We have evaluated codon usage bias in Drosophila histone genes and have obtained the nucleotide sequence of a 5,161-bp D. hydei histone gene repeat unit. This repeat contains genes for all five histone proteins (H1, H2a, H2b, H3, and H4) and differs from the previously reported one by a second EcoRI site. These D. hydei repeats have been aligned to each other and to the 5.0-kb (i.e., long) and 4.8-kb (i.e., short) histone repeat types from D. melanogaster. In each species, base composition at synonymous sites is similar to the average genomic composition and approaches that in the small intergenic spacers of the histone gene repeats. Accumulation of synonymous changes at synonymous sites after the species diverged is quite high. Both of these features are consistent with the relatively low codon usage bias observed in these genes when compared with other Drosophila genes. Thus, the generalization that abundantly expressed genes in Drosophila have high codon bias and low rates of silent substitution does not hold for the histone genes.      

GOAT induced ghrelin acylation regulates hedonic feeding

Ghrelin is an orexigenic hormone that regulates homeostatic and reward-related feeding behavior. Recent evidence indicates that acylation of ghrelin by the gut enzyme ghrelin O-acyl transferase (GOAT) is necessary to render ghrelin maximally active within its target tissues. Here we tested the hypothesis that GOAT activity modulates food motivation and food hedonics using behavioral pharmacology and mutant mice deficient for GOAT and the ghrelin receptor (GHSR). We evaluated operant responding following pharmacological administration of acyl-ghrelin and assessed the necessity of endogenous GOAT activity for operant responding in GOAT and GHSR-null mice. Hedonic-based feeding behavior also was examined in GOAT-KO and GHSR-null mice using a “Dessert Effect” protocol in which the intake of a palatable high fat diet “dessert” was assessed in calorically-sated mice. Pharmacological administration of acyl-ghrelin augmented operant responding; notably, this effect was dependent on intact GHSR signaling. GOAT-KO mice displayed attenuated operant responding and decreased hedonic feeding relative to controls. These behavioral results correlated with decreased expression of the orexin-1 receptor in reward-related brain regions in GOAT-KO mice. In summary, the ability of ghrelin to stimulate food motivation is dependent on intact GHSR signaling and modified by endogenous GOAT activity. Furthermore, GOAT activity is required for hedonic feeding behavior, an effect potentially mediated by forebrain orexin signaling. These data highlight the significance of the GOAT–ghrelin system for the mediation of food motivation and hedonic feeding.     

Predicting direct protein interactions from affinity purification mass spectrometry data

Background  

Affinity purification followed by mass spectrometry identification (AP-MS) is an increasingly popular approach to observe protein-protein interactions (PPI) in vivo. One drawback of AP-MS, however, is that it is prone to detecting indirect interactions mixed with direct physical interactions. Therefore, the ability to distinguish direct interactions from indirect ones is of much interest.     

A functional role for the p62–ERK1 axis in the control of energy homeostasis and adipogenesis

In vivo genetic inactivation of the signalling adapter p62 leads to mature-onset obesity and insulin resistance, which correlate with reduced energy expenditure (EE) and increased adipogenesis, without alterations in feeding or locomotor functions. Enhanced extracellular signal-regulated kinase (ERK) activity in adipose tissue from p62-knockout (p62^−/−) mice, and differentiating fibroblasts, suggested an important role for this kinase in the metabolic alterations of p62^−/− mice. Here, we show that genetic inactivation of ERK1 in p62^−/− mice reverses their increased adiposity and adipogenesis, lower EE and insulin resistance. These results establish genetically that p62 is a crucial regulator of ERK1 in metabolism.     

Receptor for retrograde transport in the apicomplexan parasite Toxoplasma gondii

Toxoplasma gondii and its apicomplexan relatives (such as Plasmodium falciparum, which causes malaria) are obligate intracellular parasites that rely on sequential protein release from specialized secretory organelles for invasion and multiplication within host cells. Because of the importance of these unusual membrane trafficking pathways for drug development and comparative cell biology, characterizing them is essential. In particular, it is unclear what role retrieval mechanisms play in parasite membrane trafficking or where they operate. Previously, we showed that T. gondii's beta-COP (TgBetaCOP; a subunit of coatomer protein complex I, COPI) and retrieval reporters localize exclusively to the zone between the parasite endoplasmic reticulum (ER) and Golgi apparatus. This suggested the existence of an HDEL receptor in T. gondii. We have now identified, cloned, and sequenced this receptor, TgERD2. TgERD2 localizes in a Golgi or ER pattern suggestive of the HDEL retrieval reporter (K. M. Hager, B. Striepen, L. G. Tilney, and D. S. Roos, J. Cell Sci. 112:2631-2638, 1999). A functional assay reveals that TgERD2 is able to complement the Saccharomyces cerevisiae ERD2 null mutant. Retrieval studies reveal that stable expression of a fluorescent exogenous retrieval ligand results in a dispersal of betaCOP signal throughout the cytoplasm and, surprisingly, results in betaCOP staining of the vacuolar space of the parasite. In contrast, stable expression of TgERD2GFP does not appear to disturb betaCOP staining. In addition to TgERD2, Toxoplasma contains two more divergent ERD2 relatives. Phylogenetic analysis reveals that these proteins belong to a previously unrecognized ERD2 subfamily common to plants and alveolate organisms and as such could represent mediators of parasite-specific retrieval functions. No evidence of class 2 ERD2 proteins was found in metazoan organisms or fungi.     

Structure detection through automated covariance search

This paper summarizes our investigations into the computationaldetection of secondary and tertiary structure of ribosomal RNA.We have developed a new automated procedure that not only identifiespotential secondary and tertiary structural interactions, butalso provides the covariation evidence that supports the proposedbondings, and any counterevidence that can be detected in theknown sequences. A small number of previously unknown higher-orderstructural features have been detected in individual RNA molecules(16S rRNA and 7S RNA) through the use of our automated procedure.We are systematically studying mitochondrial rRNA, seeking tertiarystructure within 16S rRNA and quaternary structure between J6Sand23S rRNA. To test hypotheses suggested by an examination ofour program's output, our colleagues in biology are sequencingkey portions of the 23S ribosomal RNA for species in which theknown 16S ribosomal RNA exhibits variation (from the dominantpattern) at the site of a proposed bonding. Our ultimate hopeis that automated covariation analysis will contribute significantlyto a refined picture of ribosomal structure. Received on January 17, 1990; accepted on June 1, 1990     

Molecular evolution of the vesicle coat component betaCOP in Toxoplasma gondii

Coatomer coated (COPI) vesicles play a pivotal role for multiple membrane trafficking steps throughout the eukaryotic cell. Our focus is on betaCOP, one of the most well known components of the COPI multi-protein complex. Amino acid differences in betaCOP may dictate functional divergence across species during the course of evolution, especially with regards to the evolutionary pressures on obligate intracellular parasites. A bioinformatic analysis of betaCOP amino acid sequences was conducted for 49 eukaryotic species. Cloning and sequence analysis of the Toxoplasma gondii betaCOP homologue revealed several amino acid insertions unique to T. gondii and one C-terminal insertion that is unique to apicomplexan parasites. These findings led us to investigate the possibility that betaCOP experienced functional divergence during the course of its evolution. Bayesian phylogenetic analysis revealed a tree consistent with pan eukaryote distribution and long-branch lengths were observed among the apicomplexans. Further analysis revealed that kinetoplast betaCOP underwent the most amount of change, leading to perhaps an overall change of function. In comparison, T. gondii exhibited subtle yet specific amino acid changes. The amino acid substitutions did not occur in the same places as other lineages, suggesting that TgbetaCOP has a role specific to the apicomplexans. Our work identifies 48 residues that are likely to be functionally important when comparing apicomplexan, kinetoplastid, and fungal betaCOP.