Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.     

The Golgi-Localized γ-Ear-Containing ARF-Binding (GGA) Proteins Alter Amyloid-β Precursor Protein (APP) Processing through Interaction of Their GAE Domain with the Beta-Site APP Cleaving Enzyme 1 (BACE1)

Proteolytic processing of amyloid-β precursor protein (APP) by beta-site APP cleaving enzyme 1 (BACE1) is the initial step in the production of amyloid beta (Aβ), which accumulates in senile plaques in Alzheimer’s disease (AD). Essential for this cleavage is the transport and sorting of both proteins through endosomal/Golgi compartments. Golgi-localized γ-ear-containing ARF-binding (GGA) proteins have striking cargo-sorting functions in these pathways. Recently, GGA1 and GGA3 were shown to interact with BACE1, to be expressed in neurons, and to be decreased in AD brain, whereas little is known about GGA2. Since GGA1 impacts Aβ generation by confining APP to the Golgi and perinuclear compartments, we tested whether all GGAs modulate BACE1 and APP transport and processing. We observed decreased levels of secreted APP alpha (sAPPα), sAPPβ, and Aβ upon GGA overexpression, which could be reverted by knockdown. GGA-BACE1 co-immunoprecipitation was impaired upon GGA-GAE but not VHS domain deletion. Autoinhibition of the GGA1-VHS domain was irrelevant for BACE1 interaction. Our data suggest that all three GGAs affect APP processing via the GGA-GAE domain.     

Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain

Amyloid-related diseases are a group of illnesses in which an abnormal accumulation of proteins into fibrillar structures is evident. Results from a wide range of studies, ranging from identification of amyloid-β dimers in the brain to biophysical characterization of the interactions between amyloidogenic peptides and lipid membranes during fibril growth shed light on the initial events which take place during amyloid aggregation. Accounts of fibril disaggregation and formation of globular aggregates due to interactions with lipids or fatty acids further demonstrate the complexity of the aggregation process and the difficulty to treat amyloid-related diseases. There is an inherent difficulty in generalizing from studies of aggregation in vitro, but the involvement of too many cellular components limits the ability to follow amyloid aggregation in a cellular (or extracellular) context. Fortunately, the development of experimental methods to generate stable globular aggregates suggests new means of studying the molecular events associated with amyloid aggregation. Furthermore, simulation studies enable deeper understanding of the experimental results and provide useful predictions that can be tested in the laboratory. Computer simulations can nowadays provide molecular or even atomistic details that are experimentally not available or very difficult to obtain. In the present review, recent developments on modelling and experiments of amyloid aggregation are reviewed, and an integrative account on how isolated interactions (as observed in vitro and in silico) combine during the course of amyloid-related diseases is presented. Finally, it is argued that an integrative approach is necessary to get a better understanding of the protein aggregation process.     

Evaluation of CuO oxidation parameters for determining the source and stage of lignin degradation in soil

The composition of phenols and other aromatic compounds in organic and mineral soil horizons and their respective source vegetation from different climatic zones of the Canadian Prairies were analyzed using CuO oxidation and gas chromatography-mass spectrometry (GC-MS) to investigate the stage of lignin degradation. Parameters based on the CuO oxidation products were calculated for the soils and corresponding vegetation to determine the lignin sources and to monitor the lignin degradation. In addition to the widely used lignin monomer parameters, parameters resulting from lignin-derived phenolic dimers are used for the first time to assess lignin degradation in soils. The composition of lignin-derived phenols (S/V, C/V) in soil closely matches the composition observed in their respective source plants (grass, Aspen, Pine) reflecting the preservation of characteristic lignin patterns in soils. Degradation parameters based on lignin phenols and benzenes derived from tannins or other phenolic biomolecules indicate a progressive degradation from the vegetation to the soil horizons. In addition to commonly used lignin monomer indicators, parameters based on the lignin dimers are applied. Lignin degradation is found to be lowest in the Pine forest, intermediate in the grassland soils and highest in the Aspen-grassland transition soil. Degradation parameters based on non-lignin aromatic derivatives (3,5-dihydroxybenzoic acid, benzenepolycarboxylic acids) demonstrate a similar trend. The lignin from samples in the cooler climate (Black Chernozems) is observed to be more oxidized than in the soils from the warmer climate (Brown Chernozems) suggesting that abiotic processes may be in involved in the alteration of lignin and other phenolic biomolecules in soils. The results indicate that the comparative analysis of CuO oxidation products of soils and source vegetation is a valuable tool to assess the sources and degradation of lignin in soils.     

Localization of vacuolar transport receptors and cargo proteins in the Golgi apparatus of developing Arabidopsis embryos

Using immunogold electron microscopy, we have investigated the relative distribution of two types of vacuolar sorting receptors (VSR) and two different types of lumenal cargo proteins, which are potential ligands for these receptors in the secretory pathway of developing Arabidopsis embryos. Interestingly, both cargo proteins are deposited in the protein storage vacuole, which is the only vacuole present during the bent-cotyledon stage of embryo development. Cruciferin and aleurain do not share the same pattern of distribution in the Golgi apparatus. Cruciferin is mainly detected in the cis and medial cisternae, especially at the rims where storage proteins aggregate into dense vesicles (DVs). Aleurain is found throughout the Golgi stack, particularly in the trans cisternae and trans Golgi network where clathrin-coated vesicles (CCVs) are formed. Nevertheless, aleurain was detected in both DV and CCV. VSR-At1, a VSR that recognizes N-terminal vacuolar sorting determinants (VSDs) of the NPIR type, localizes mainly to the trans Golgi and is hardly detectable in DV. Receptor homology-transmembrane-RING H2 domain (RMR), a VSR that recognizes C-terminal VSDs, has a distribution that is very similar to that of cruciferin and is found in DV. Our results do not support a role for VSR-At1 in storage protein sorting, instead RMR proteins because of their distribution similar to that of cruciferin in the Golgi apparatus and their presence in DV are more likely candidates. Aleurain, which has an NPIR motif and seems to be primarily sorted via VSR-At1 into CCV, also possesses putative hydrophobic sorting determinants at its C-terminus that could allow the additional incorporation of this protein into DV.     

Esr,a second locus in the house mouse controlling esterase-5

Electrophoretic variation characterized by the presence (ES-5B⁺) or absence (ES-5B^–) of esterase-5B in the plasma of the house mouse has been observed. It is suggested that the expression of esterase-5B is controlled by an autosomal locus, Esr, linked to Ldr-1 on chromosome 6, in addition to the presumptive structural locus Es-5, which is located on chromosome 8. A gene order of Lyt-3-Esr-Ldr-1 was determined by two crosses.Supported by the Deutsche Forschungsgemeinschaft (SFB 46).This is communication No. 33 of a research program devoted to the investigation of cellular distribution and genetics of nonspecific esterases.     

Solid-Supported Oligonucleotide Systems for Special Biomedical Applications

Abstract

The use of composite beads consisting of a 6 μm polystyrene core with 30 nm surface-bound silica particles to routine automatic oligodeoxynucleotide (ODN) synthesis is described.     

Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.