Retrograde Traffic Out of the Yeast Vacuole to the TGN Occurs via the Prevacuolar/Endosomal Compartment

A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid–containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.     

Extracellular enzyme localization during interspecific fungal interactions

Abstract Confronted colonies of Phlebia radiata, P. rufa, Coriolus versicolor, Stereum hirsutum, Phanerochaete velutina and Hypholoma fasciculare showed spatially and temporally heterogeneous patterns of loccase-α-naphthol and peroxidase activities. These activities were coincident in axenic cultures. but were not always so during interaction. Confrontation between species resulted in induction of phenoloxidase activities, even within coenocytic colony regions of Phlebia species which were normally void of such activities in axenic culture. These events resulted in restriction of C. versicolor growth during interaction with P. rufa.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Non-destructive hydrocarbon extraction from Botryococcus braunii BOT-22 (race B)

There is worldwide interest in developing algal biofuel. One main reason for the lack of success so far in producing a sustainable transport fuel from microalgae is the high cost of biomass processing, especially dewatering and oil extraction. There is also a significant cost involved in the energy content of the nutrient fertilisers required for biomass production. Non-destructive oil extraction or “milking” from algae biomass has the potential to bypass all of these hurdles. Using a “milking” strategy means that there would be no need for (a) biomass dewatering, (b) breaking cells for oil extraction and (c) addition of nutrients to the culture, resulting in a significant reduction in energy and fertiliser cost involved in production of biofuel from algae. We make use of the natural tendency of Botryococcus to produce external hydrocarbon in the extracellular matrix. In current study, we showed that external hydrocarbon from Botryococcus braunii BOT-22 can be non-destructively extracted using n-heptane (optimum contact time with n-heptane?=?20 min). We were able to recover almost the entire de novo-produced external hydrocarbons at 5- and 11-day intervals when the culture was maintained with or without 1 % CO₂ addition, respectively. This repeated non-destructive extraction of external hydrocarbon of B. braunii was possible for up to 70 days when 1 % CO₂ was supplied to the culture. When CO₂ was limited, a 70 % lower external hydrocarbon productivity was achieved using the same process. Although the productivity of external hydrocarbon of 9.33 mg L^?1 day^?1 of the “milked” culture is low in these un-optimised cultures, it was 1.3?±?0.2-fold higher compared with that of a conventional semicontinuous culture, showing the potential of this method.     

Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers

In this study, the diversity of Plasmodium vivax populations circulating in Pakistan and Iran has been investigated by using circumsporozoite protein (csp) and merozoite surface proteins 1 and 3α (msp-1 and msp-3α) genes as genetic markers. Infected P. vivax blood samples were collected from Pakistan (n = 187) and Iran (n = 150) during April to October 2008, and were analyzed using nested-PCR/RFLP and sequencing methods. Genotyping pvmsp-1 (variable block 5) revealed the presence of type 1, type 2 and recombinant type 3 allelic variants, with type 1 predominant, in both study areas. The sequence analysis of 33 P. vivax isolates from Pakistan and 30 from Iran identified 16 distinct alleles each, with one allele (R-8) from Iran which was not reported previously. Genotyping pvcsp gene also showed that VK210 type is predominant in both countries. Moreover, based on the size of amplified fragment of pvmsp-3α, three major types: type A (1800 bp), type B (1500 bp) and type C (1200 bp), were distinguished among the examined isolates that type A was predominant among Pakistani (72.7%) and Iranian (77.3%) parasites. PCR/RFLP products of pvmsp-3α with HhaI and AluI have detected 40 and 39 distinct variants among Pakistani and Iranian examined isolates, respectively. Based on these three studied genes, the rate of combined multiple genotypes were 30% and 24.6% for Pakistani and Iranian P. vivax isolates, respectively. These results indicate an extensive diversity in the P. vivax populations in both studies.     

Pierce into the Native Structure of Ata,a Trimeric Autotransporter of Acinetobacter baumannii ATCC 17978

International Journal of Peptide Research and Therapeutics - Acinetobacter baumannii is an important pathogen responsible for nosocomial infections worldwide. Trimeric autotransporters, the...     

Arabidopsis Sec1/Munc18 Protein SEC11 Is a Competitive and Dynamic Modulator of SNARE Binding and SYP121-Dependent Vesicle Traffic

The Arabidopsis thaliana Qa-SNARE SYP121 (=SYR1/PEN1) drives vesicle traffic at the plasma membrane of cells throughout the vegetative plant. It facilitates responses to drought, to the water stress hormone abscisic acid, and to pathogen attack, and it is essential for recovery from so-called programmed stomatal closure. How SYP121-mediated traffic is regulated is largely unknown, although it is thought to depend on formation of a fusion-competent SNARE core complex with the cognate partners VAMP721 and SNAP33. Like SYP121, the Arabidopsis Sec1/Munc18 protein SEC11 (=KEULE) is expressed throughout the vegetative plant. We find that SEC11 binds directly with SYP121 both in vitro and in vivo to affect secretory traffic. Binding occurs through two distinct modes, one requiring only SEC11 and SYP121 and the second dependent on assembly of a complex with VAMP721 and SNAP33. SEC11 competes dynamically for SYP121 binding with SNAP33 and VAMP721, and this competition is predicated by SEC11 association with the N terminus of SYP121. These and additional data are consistent with a model in which SYP121-mediated vesicle fusion is regulated by an unusual “handshaking” mechanism of concerted SEC11 debinding and rebinding. They also implicate one or more factors that alter or disrupt SEC11 association with the SYP121 N terminus as an early step initiating SNARE complex formation.     

Biomonitoring of tobacco smoke carcinogenicity by dosimetry of DNA adducts and genotyping and phenotyping of biotransformational enzymes: a review on polycyclic aromatic hydrocarbons

In this review article, we summarize the data on tobacco smoke carcinogenicity in relation to DNA adduct dosimetry and genotyping and phenotyping of biotransformational enzymes. A major class of carcinogens, polycyclic aromatic hydrocarbons, present in substantial quantities in tobacco smoke, is discussed. The historical background and an overview of the metabolic pathways are given. The epidemiological and biological data in particular on dosimetry of the representative DNA adducts and genotyping and phenotyping of the respective activating and detoxifying enzymes are presented. The salient findings are highlighted, the uncertainties are underlined and, finally, recommendations for future research are made.     

Time-resolved fluorescence study of excitation energy transfer in the cyanobacterium Anabaena PCC 7120

Photosynthesis Research - Excitation energy transfer (EET) and trapping in Anabaena variabilis (PCC 7120) intact cells, isolated phycobilisomes (PBS) and photosystem I (PSI) complexes have been...     

Decellularized amniotic membrane Scaffolds improve differentiation of iPSCs to functional hepatocyte-like cells

Human-induced pluripotent stem cells-derived hepatocyte-like cells (hiPSCs-HLCs) holds considerable promise for future clinical personalized therapy of liver disease. However, the low engraftment of these cells in the damaged liver microenvironment is still an obstacle for potential application. In this study, we explored the effectiveness of decellularized amniotic membrane (dAM) matrices for culturing of iPSCs and promoting their differentiation into HLCs. The DNA content assay and histological evaluation indicated that cellular and nuclear residues were efficiently eliminated and the AM extracellular matrix component was maintained during decelluarization. DAM matrices were developed as three-dimensional scaffolds and hiPSCs were seeded into these scaffolds in defined induction media. In dAM scaffolds, hiPSCs-HLCs gradually took a typical shape of hepatocytes (polygonal morphology). HiPSCs-HLCs that were cultured into dAM scaffolds showed a higher level of hepatic markers than those cultured in tissue culture plates (TCPs). Moreover, functional activities in term of albumin and urea synthesis and CYP3A activity were significantly higher in dAM scaffolds than TCPs over the same differentiation period. Thus, based on our results, dAM scaffold might have a considerable potential in liver tissue engineering, because it can improve hepatic differentiation of hiPSCs which exhibited higher level of the hepatic marker and more stable metabolic functions.