mTrs130 Is a Component of a Mammalian TRAPPII Complex,a Rab1 GEF That Binds to COPI-coated Vesicles

The GTPase Rab1 regulates endoplasmic reticulum-Golgi and early Golgi traffic. The guanine nucleotide exchange factor (GEF) or factors that activate Rab1 at these stages of the secretory pathway are currently unknown. Trs130p is a subunit of the yeast TRAPPII (transport protein particle II) complex, a multisubunit tethering complex that is a GEF for the Rab1 homologue Ypt1p. Here, we show that mammalian Trs130 (mTrs130) is a component of an analogous TRAPP complex in mammalian cells, and we describe for the first time the role that this complex plays in membrane traffic. mTRAPPII is enriched on COPI (Coat Protein I)-coated vesicles and buds, but not Golgi cisternae, and it specifically activates Rab1. In addition, we find that mTRAPPII binds to γ1COP, a COPI coat adaptor subunit. The depletion of mTrs130 by short hairpin RNA leads to an increase of vesicles in the vicinity of the Golgi and the accumulation of cargo in an early Golgi compartment. We propose that mTRAPPII is a Rab1 GEF that tethers COPI-coated vesicles to early Golgi membranes.     

Subdomain-specific localization of CLIMP-63 (p63) in the endoplasmic reticulum is mediated by its luminal alpha-helical segment

The microtubule-binding integral 63 kD cytoskeleton-linking membrane protein (CLIMP-63; former name, p63) of the rough endoplasmic reticulum (ER) is excluded from the nuclear envelope. We studied the mechanism underlying this ER subdomain-specific localization by mutagenesis and structural analysis. Deleting the luminal but not cytosolic segment of CLIMP-63 abrogated subdomain-specific localization, as visualized by confocal microscopy in living cells and by immunoelectron microscopy using ultrathin cryosections. Photobleaching/recovery analysis revealed that the luminal segment determines restricted diffusion and immobility of the protein. The recombinant full-length luminal segment of CLIMP-63 formed alpha-helical 91-nm long rod-like structures as evident by circular dichroism spectroscopy and electron microscopy. In the analytical ultracentrifuge, the luminal segment sedimented at 25.7 S, indicating large complexes. The complexes most likely arose by electrostatic interactions of individual highly charged coiled coils. The findings indicate that the luminal segment of CLIMP-63 is necessary and sufficient for oligomerization into alpha-helical complexes that prevent nuclear envelope localization. Concentration of CLIMP-63 into patches may enhance microtubule binding on the cytosolic side and contribute to ER morphology by the formation of a protein scaffold in the lumen of the ER.     

Toward a low-carbon and circular building sector: Building strategies and urbanization pathways for the Netherlands

Buildings are an important part of society's environmental impacts, both in the construction and in the use phase. As the energy performance of buildings improve, construction materials become more important as a cause of environmental impact. Less attention has been given to those materials. We explore, as an alternative for conventional buildings, the use of biobased materials and circular building practices. In addition to building design, we analyze the effect of urbanization. We assess the potential to close material cycles together with the material related impact, between 2018 and 2050 in the Netherlands. Our results show a limited potential to close material cycles until 2050, as a result of slow stock turnover and growth of the building stock. At present, end-of-life recycling rates are low, further limiting circularity. Primary material demand can be lowered when shifting toward biobased or circular construction. This shift also reduces material related carbon emissions. Large-scale implementation of biobased construction, however, drastically increases land area required for wood production. Material demand differs strongly spatially and depends on the degree of urbanization. Urbanization results in higher building replacement rates, but constructed dwellings are generally small compared to scenarios with more rural developments. The approach presented in this work can be used to analyze strategies aimed at closing material cycles in the building sector and lowering buildings' embodied environmental impact, at different spatial scales.     

Organization of fusicoccin receptor in higher plant plasma membrane: relationship between affinity and molecular mass

Higher plant plasma membranes carry receptors of different affinity for the phytotoxin fusicoccin. Reception of fusicoccin involves proteins belonging to the highly conserved 14-3-3 family, but the complete structure of the fusicoccin receptor (FCR) is unknown. Using radiation inactivation analysis, we estimated the molecular masses of low-affinity and high-affinity FCR at 63 +/- 7 and 130 +/- 15 kD, respectively. The dose dependences of receptor inactivation indicate that microsomal specimens contain "silent" FCRs of 420 +/- 90 kD in amounts commensurate with that of the active FCRs. Both low- and high-affinity FCRs are inactivated by hydrolytic enzymes from the outer surface of the plasma membrane, and impairment of protoplast integrity causes an irreversible transition of the low-affinity binding site into the high-affinity one. A scheme is proposed for the organization of different types of FCR in the plasma membrane, implying that the membrane affinity for fusicoccin reflects the interaction between proteins in the FCR complex.     

NaCl-preferring NZB/B1NJ mice and NaCl-avoiding CBA/J mice have similar amiloride inhibition of chorda tympani responses to NaCl

Integrated chorda tympani nerve responses to NaCl were studied in two mouse strains, an NaCl-preferring NZB/B1NJ and an NaCl-avoiding CBA/J. The NaCl responses of both strains had similar magnitude and were suppressed by amiloride to a similar extent. This suggests that peripheral gustatory responsiveness to NaCl is not the only mechanism underlying mouse strain variation in NaCl acceptance.      

Genetics of alcohol dehydrogenase and aldehyde dehydrogenase from Monodelphis domestica cornea: further evidence for identity of corneal aldehyde dehydrogenase with a major soluble protein

A didelphid marsupial, the gray short-tailed opossum (Monodelphis domestica), was used as a model species to study the biochemical genetics of alcohol dehydrogenases (ADHs) and aldehyde dehydrogenase (ALDH) in corneal tissue. Isoelectric point variants of corneal ALDH (designated ALDH3) and a major soluble protein in corneal extracts were observed among eight families of animals used in studying the genetics of these proteins. Both phenotypes exhibited identical patterns following PAGE-IEF and were inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal ALDH with this major soluble protein, and supported biochemical evidence, recently reported for purified bovine corneal ALDH, that this enzyme constitutes a major portion of soluble corneal protein (Abedinia et al. 1990). Isoelectric point variants for corneal ADH were also observed, with patterns for the two major forms (ADH3 and ADH4) and one minor form (ADH5) being consistent with the presence of two ADH subunits (designated gamma and delta), and variant phenotypes existing for the gamma subunit. The genetics of this enzyme was studied in the eight families, and the results were consistent with codominant expression of two alleles at a single locus (designated ADH3). It is relevant that a major detoxification function has been proposed for corneal ADH and ALDH, in the oxidoreduction of peroxidic aldehydes induced by available oxygen and UV-B light (Holmes & VandeBerg, 1986a). In addition, a direct role for corneal ALDH as a UV-B photoreceptor in this anterior eye tissue has also been proposed (Abedinia et al. 1990).     

INDUCTION OF ERYTHROID MATURATION BY DIMETHYL SULFOXIDE IN FRIEND LEUKEMIC CELLS

Enhancement of the erythroid maturation in Friend virus-induced leukemic cells has been examined in vitro by the treatment with dimethyl sulfoxide (DMSO). Although the cell growth was inhibited in the medium containing 2% DMSO, many cells remained viable for a week. By the 3rd day of the culture, the cells treated with DMSO became more strongly agglutinated by phytohemagglutinin than the cells incubated without DMSO. Mouse erythrocyte membrane-specific antigens were also detectable at the 4th day. At the 8th day of the culture hemoglobin synthesis was apparently demonstrated in the cells treated with DMSO, which could not be seen in the untreated cells. Maturation or differentiation along the erythroid pathway in Friend leukemic cells by DMSO is discussed on these markers.     

Antiquity and diversity of aboriginal Australian Y‐chromosomes

Humans are limited in their capacity to convert protein into energy. We present a hypothesis that a “bell” shaped thorax and a wide pelvis evolved in Neandertals, at least in part, as an adaptation to a high protein diet. A high protein diet created a need to house an enlarged liver and urinary system in a wider lower trunk. To test the hypothesis, we applied a model developed to identify points of nutritional stress. A ratio of obligatory dietary fat to total animal fat and protein sourced calories is calculated based on various known and estimated parameters. Stress is identified when the obligatory dietary fat ratio is higher than fat content ratios in available prey. The model predicts that during glacial winters, when carbohydrates weren't available, 74%?85% of Neandertals' caloric intake would have had to come from animal fat. Large animals contain around 50% fat calories, and their fat content is diminished during winter, so a significant stressful dietary fat deficit was identified by the model. This deficit could potentially be ameliorated by an increased capability to convert protein into energy. Given that high protein consumption is associated with larger liver and kidneys in animal models, it appears likely that the enlarged inferior section of the Neandertals thorax and possibly, in part, also his wide pelvis, represented an adaptation to provide encasement for those enlarged organs. Behavioral and evolutionary implications of the hypothesis are also discussed. Am J Phys Anthropol 160:367–378, 2016. © 2016 Wiley Periodicals, Inc.     

Selective, high-resolution fluorescence imaging of mitochondrial Ca2+ concentration.

We have developed a digital image processing technique based on highpass filtering of microfluorimetric images for selective transmission of fine image details corresponding to mitochondria. This technique enabled the detection of the mitochondrial calcium signals with high selectivity, simultaneously with the cytosolic calcium signal. The validity of this technique was supported in primary cultures of rat brain capillary endothelial cells loaded with X-rhod-1 by the results that (i) inhibition of the mitochondrial Ca2+ uptake by discharging the mitochondrial membrane potential selectively abolished the transient of the highpass filtered signal evoked by ATP, and (ii) CGP-37157, a selective blocker of the mitochondrial Na+/Ca2+ exchanger, increased the peak amplitude of highpass filtered (mitochondrial) Ca2+ transients and caused a sustained plateau. The highpass filtering technique enabled the analysis of the mitochondrial Ca2+ transients in high temporal resolution. We found a uniform and monophasic rise of [Ca2+] in the mitochondrial population of the cell, following the cytosolic [Ca2+] with a delay at onset and peak. The introduced highpass filtering technique is a powerful tool in the high spatial and temporal resolution analysis of mitochondrial calcium transients, and it could be especially important in specimens where genetically targeted probes fail.     

S6 kinase inactivation impairs growth and translational target phosphorylation in muscle cells maintaining proper regulation of protein turnover

A defect in protein turnover underlies multiple forms of cell atrophy. Since S6 kinase (S6K)-deficient cells are small and display a blunted response to nutrient and growth factor availability, we have hypothesized that mutant cell atrophy may be triggered by a change in global protein synthesis. By using mouse genetics and pharmacological inhibitors targeting the mammalian target of rapamycin (mTOR)/S6K pathway, here we evaluate the control of translational target phosphorylation and protein turnover by the mTOR/S6K pathway in skeletal muscle and liver tissues. The phosphorylation of ribosomal protein S6 (rpS6), eukaryotic initiation factor-4B (eIF4B), and eukaryotic elongation factor-2 (eEF2) is predominantly regulated by mTOR in muscle cells. Conversely, in liver, the MAPK and phosphatidylinositol 3-kinase pathways also play an important role, suggesting a tissue-specific control. S6K deletion in muscle mimics the effect of the mTOR inhibitor rapamycin on rpS6 and eIF4B phosphorylation without affecting eEF2 phosphorylation. To gain insight on the functional consequences of these modifications, methionine incorporation and polysomal distribution were assessed in muscle cells. Rates and rapamycin sensitivity of global translation initiation are not altered in S6K-deficient muscle cells. In addition, two major pathways of protein degradation, autophagy and expression of the muscle-specific atrophy-related E3 ubiquitin ligases, are not affected by S6K deletion. Our results do not support a role for global translational control in the growth defect due to S6K deletion, suggesting specific modes of growth control and translational target regulation downstream of mTOR. signal transduction; atrophy; autophagy