A conserved cysteine residue of Pichia pastoris Pex20p is essential for its recycling from the peroxisome to the cytosol

We identified a cysteine residue, conserved near the N terminus of Pex5p- and Pex20p-like proteins, that is essential for the cytosolic relocation of peroxisomal Pex20p. Surprisingly, this residue is not completely essential for the function of the protein; its point mutation into a serine in Pex20p(C8S) causes the accumulation of the protein at the peroxisome membrane, but this is quickly followed by its subsequent degradation by an ubiquitin-dependent quality control pathway called RADAR (receptor accumulation and degradation in the absence of recycling). This degradative pathway allows partial growth of the Pex20p(C8S) mutant on peroxisome-requiring medium. Mutation of cysteine 8 (C8S) and lysine 19 (K19R), the target residue of the RADAR pathway within Pex20p, leads to a stable but non-functional protein because it fails to recycle to the cytosol. This suggests a role for Cys-8 in Pex20p recycling and that constitutive degradation of peroxisomal receptors can be a partially functional alternative to receptor recycling. In addition, expression of this mutant protein in wild-type cells confers a dominant-negative, oleate-specific growth defect, which is a useful tool for a better understanding of peroxisomal receptor recycling.     

Peroxisomal Pex3 Activates Selective Autophagy of Peroxisomes via Interaction with the Pexophagy Receptor Atg30

Pexophagy is a process that selectively degrades peroxisomes by autophagy. The Pichia pastoris pexophagy receptor Atg30 is recruited to peroxisomes under peroxisome proliferation conditions. During pexophagy, Atg30 undergoes phosphorylation, a prerequisite for its interactions with the autophagy scaffold protein Atg11 and the ubiquitin-like protein Atg8. Atg30 is subsequently shuttled to the vacuole along with the targeted peroxisome for degradation. Here, we defined the binding site for Atg30 on the peroxisomal membrane protein Pex3 and uncovered a role for Pex3 in the activation of Atg30 via phosphorylation and in the recruitment of Atg11 to the receptor protein complex. Pex3 is classically a docking protein for other proteins that affect peroxisome biogenesis, division, and segregation. We conclude that Pex3 has a role beyond simple docking of Atg30 and that its interaction with Atg30 regulates pexophagy in the yeast P. pastoris.     

Distinct Actions of Rab3 and Rab27 GTPases on Late Stages of Exocytosis of Insulin

Rab GTPases associated with insulin‐containing secretory granules (SGs) are key in targeting, docking and assembly of molecular complexes governing pancreatic β‐cell exocytosis. Four Rab3 isoforms along with Rab27A are associated with insulin granules, yet elucidation of the distinct roles of these Rab families on exocytosis remains unclear. To define specific actions of these Rab families we employ Rab3GAP and/or EPI64A GTPase‐activating protein overexpression in β‐cells from wild‐type or Ashen mice to selectively transit the entire Rab3 family or Rab27A to a GDP‐bound state. Ashen mice carry a spontaneous mutation that eliminates Rab27A expression. Using membrane capacitance measurements we find that GTP/GDP nucleotide cycling of Rab27A is essential for generation of the functionally defined immediately releasable pool (IRP) and central to regulating the size of the readily releasable pool (RRP). By comparison, nucleotide cycling of Rab3 GTPases, but not of Rab27A, is essential for a kinetically rapid filling of the RRP with SGs. Aside from these distinct functions, Rab3 and Rab27A GTPases demonstrate considerable functional overlap in building the readily releasable granule pool. Hence, while Rab3 and Rab27A cooperate to generate release‐ready SGs in β‐cells, they also direct unique kinetic and functional properties of the exocytotic pathway.      

Biological Differences between Brackish and Fresh Water-Derived Aedes aegypti from Two Locations in the Jaffna Peninsula of Sri Lanka and the Implications for Arboviral Disease Transmission

The mainly fresh water arboviral vector Aedes aegypti L. (Diptera: Culicidae) can also undergo pre-imaginal development in brackish water of up to 15 ppt (parts per thousand) salt in coastal areas. We investigated differences in salinity tolerance, egg laying preference, egg hatching and larval development times and resistance to common insecticides in Ae. aegypti collected from brackish and fresh water habitats in Jaffna, Sri Lanka. Brackish water-derived Ae. aegypti were more tolerant of salinity than fresh water-derived Ae. aegypti and this difference was only partly reduced after their transfer to fresh water for up to five generations. Brackish water-derived Ae. aegypti did not significantly discriminate between 10 ppt salt brackish water and fresh water for oviposition, while fresh water-derived Ae. aegypti preferred fresh water. The hatching of eggs from both brackish and fresh water-derived Ae. aegypti was less efficient and the time taken for larvae to develop into pupae was prolonged in 10 ppt salt brackish water. Ae. aegypti isolated from coastal brackish water were less resistant to the organophosphate insecticide malathion than inland fresh water Ae. aegypti. Brackish and fresh water-derived Ae. aegypti however were able to mate and produce viable offspring in the laboratory. The results suggest that development in brackish water is characterised by pertinent biological changes, and that there is restricted genetic exchange between coastal brackish and inland fresh water Ae. aegypti isolates from sites 5 km apart. The findings highlight the need for monitoring Ae. aegypti developing in coastal brackish waters and extending vector control measures to their habitats.     

Whole cell nuclear magnetic resonance characterization of two photochemically active states of the photosynthetic reaction center in heliobacteria

Applying photo-CIDNP (photochemically induced dynamic nuclear polarization) MAS (magic-angle spinning) nuclear magnetic resonance to whole cells of Heliobacillus (Hb.) mobilis, we demonstrate that heliobacterial reaction centers are operational in two different states as indicated by the occurrence of a light-induced spin-correlated radical pair. A culture maintained anaerobically is called "Braunstoff" (German for "brown substance"). After exposure to oxygen, Braunstoff is converted to "Grünstoff" ("green substance") as indicated by a color change due to the conversion of BChl g to Chl a(F). It is shown that electron transfer occurs symmetrically via both branches of cofactors in both forms. The donor and acceptor cofactors remain identical and unchanged upon conversion, while the intermediate accessory cofactors are transformed from BChl g to Chl a(F). The donor triplet state in Braunstoff is localized on the special pair donor and lives for 100 μs, demonstrating the absence of nearby carotenoids. In Grünstoff, the donor triplet becomes mobile and appears to be formed on an accessory cofactor.     

Equilibrium binding and kinetic characterization of putative tetracycline repressor family transcription regulator Fad35R from Mycobacterium tuberculosis

Fatty acids play critical role in the survival and virulence of Mycobacterium?tuberculosis (Mtb). Activation of fatty acids by acyl-CoA synthetases (Fad) into fatty acyl-CoA is the first and one of the crucial steps in fatty acid metabolism. Mtb possesses 36 fatty acyl-CoA synthetases, unlike Escherichia?coli, which has single enzyme. However, the mechanisms by which the expression of these multiple Fad genes is regulated remain uncharacterized. We characterized the DNA- and ligand-binding properties of a putative tetracycline repressor family regulator, named Fad35R, located upstream of the Fad35 gene and ScoA-citE operon. We identified a palindromic regulatory motif upstream of Fad35 and characterized the binding of Fad35R to this motif. Equilibrium binding studies show that Fad35R binds to this motif with high affinity (K(d) ～?0.033?μm) and the specificity of binding was confirmed by an electromobility gel shift assay. Kinetic studies indicate that faster association (k(a,avg) ～?5.4?×?10(4) m(-1) ·s(-1) ) and slower dissociation rates (k(d,avg) ～?5.84?×?10(-4) s(-1) ) confer higher affinity. The affinity for the promoter is maximum at 300?mm NaCl but decreases rapidly beyond this range. Ligand-binding studies indicate that Fad35R binds specifically to tetracycline and also binds to fatty acid derivatives. The promoter-binding affinity is decreased significantly in the presence of palmityl-CoA, suggesting that Fad35R can sense the levels of activated fatty acids and alter its DNA-binding activity. Our results suggest that Fad35R may be the functional homologue of FadR and controls the expression of genes in a metabolite-dependent manner. Structured digital abstract ? Fad35R?binds to?palindromic sequence?shown by surface plasmon resonance ? Fad35R?binds to?tetracycline?and?activated fatty acids?as shown by fluorescence spectroscopy.     

β-sheet topology prediction with high precision and recall for β and mixed α/β proteins

The prediction of the correct β-sheet topology for pure β and mixed α/β proteins is a critical intermediate step toward the three dimensional protein structure prediction. The predicted beta sheet topology provides distance constraints between sequentially separated residues, which reduces the three dimensional search space for a protein structure prediction algorithm. Here, we present a novel mixed integer linear optimization based framework for the prediction of β-sheet topology in β and mixed α/β proteins. The objective is to maximize the total strand-to-strand contact potential of the protein. A large number of physical constraints are applied to provide biologically meaningful topology results. The formulation permits the creation of a rank-ordered list of preferred β-sheet arrangements. Finally, the generated topologies are re-ranked using a fully atomistic approach involving torsion angle dynamics and clustering. For a large, non-redundant data set of 2102 β and mixed α/β proteins with at least 3 strands taken from the PDB, the proposed approach provides the top 5 solutions with average precision and recall greater than 78%. Consistent results are obtained in the β-sheet topology prediction for blind targets provided during the CASP8 and CASP9 experiments, as well as for actual and predicted secondary structures. The β-sheet topology prediction algorithm, BeST, is available to the scientific community at http://selene.princeton.edu/BeST/.     

Self-destruction in the line of duty.

Three cellular processes, microautophagy, macroautophagy, and the cytoplasm-to-vacuole (Cvt) pathway, are involved in the cargo delivery from the cytosol to the vacuole or lysosome. Recent findings have identified Cvt19 at the receptor for specific cargo binding in the Cvt pathway.     

Occurrence and distribution of virulent strains of Vibrio parahaemolyticus in seafoods marketed from Cochin (India)

This study was aimed for the detection of Vibrio parahaemolyticus by biochemical and molecular methods in seafood samples collected from the markets of Cochin located at the southwest coast of India. A total of seventy-two V. parahaemolyticus cultures were isolated by selecting sucrose and cellobiose non-fermenting colonies. All the biochemically confirmed strains were found to have 368-bp toxR gene fragment, while an additional 24% of the samples were confirmed as V. parahaemolyticus by toxR based polymerase chain reaction (PCR) from enrichment broths. PCR based methods are used to detect tdh, trh, and orf8 genes for the identification of pathogenic and pandemic V. parahaemolyticus. Only one out of two urease positive isolates amplified the trh (500bp) gene. About 10% of the isolates showed weak haemolysis and none were found to amplify tdh (269 bp) and orf8 (746 bp) genes, thus indicating the meager incidence of pandemic strains from this area. The incidence of trh positive isolates from market samples signals towards the adoption of stringent seafood safety measures for the products meant for human consumption.