Summary Autophagy is an evolutionarily conserved membrane trafficking process. Induction of autophagy in response to nutrient limitation or cellular stress occurs by similar mechanisms in organisms from yeast to mammals. Unlike yeast, metazoan cells rely more on growth factor signaling for a wide variety of cellular activities including nutrient uptake. How growth factor availability regulates autophagy is poorly understood. Here we show that, upon growth factor limitation, the p110β catalytic subunit of the Class IA phosphoinositide 3-kinases (PI3Ks) dissociates from growth factor receptor complexes, and increases its interaction with the small GTPase Rab5. This p110β-Rab5 association maintains Rab5 in its GTP-bound state and enhances the Rab5-Vps34 interaction that promotes autophagy. p110β mutants that fail to interact with Rab5 are defective in autophagy promotion. Hence, in mammalian cells, p110β acts as a molecular sensor for growth factor availability and induces autophagy by activating a Rab5-mediated signaling cascade.

Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.

Rabex-5 targets to early endosomes and functions as a guanine nucleotide exchange factor for Rab5. Membrane targeting is critical for Rabex-5 to activate Rab5 on early endosomes in the cell. Here, we report the identification of Rab22 as a binding site on early endosomes for direct recruitment of Rabex-5 and activation of Rab5, establishing a Rab22-Rab5 signaling relay to promote early endosome fusion. Rab22 in guanosine 5-O-(3-thio)triphosphate-loaded form, but not guanosine diphosphate-loaded form, binds to the early endosomal targeting domain (residues 81-230) of Rabex-5 in pull-down assays. Rabex-5 targets to Rab22-containing early endosomes, and Rab22 knockdown by short hairpin RNA abrogates the membrane targeting of Rabex-5 in the cell. In addition, coexpression of Rab22 and Rab5 shows synergistic enlargement of early endosomes, and this synergy is dependent on Rabex-5, providing further support for the collaboration of the two Rab GTPases in regulation of endosome dynamics. This novel Rab22-Rabex-5-Rab5 cascade is functionally important for the endocytosis and degradation of epidermal growth factor.

Aims:The objective of the present study was to investigate the optimal culture requirements for mycelial growth and exopolysaccharide production by Cordyceps jiangxiensis JXPJ 0109 in submerged culture. Methods and Results: The effects of medium ingredients (i.e. carbon and nitrogen sources, and growth factor) and other culture requirements (i.e. initial pH, temperature, etc.) on the production of mycelia and exopolysaccharide were observed using a one-factor-at-a-time method. More suitable culture requirements for mycelial growth and exopolysaccharide production were proved to be maltose, glycerol, tryptone, soya bean steep powder, yeast extract, medium capacity 200 ml in a 500-ml flask, agitation rate 180 rev min )1 , seed age 4-8 days, inoculum size 2AE5-7AE5% (v/v), etc. The optimal temperatures and initial pHs for mycelial growth and exopolysaccharide production were at 26°C and pH 5 and at 28°C and pH 7, respectively, and corresponding optimal culture age were observed to be 8 and 10 days respectively. According to the primary results of the one-factor-at-a-time experiments, the optimal medium for the mycelial growth and exopolysaccharide production were obtained using an orthogonal layout method to optimize further. Herein the effects of medium ingredients on the mycelial growth of C. jiangxiensis JXPJ 0109 were in the order of yeast extract > tryptone > maltose > CaCl 2 > glycerol > MgSO 4 > KH 2 PO 4 and the optimal concentration of each composition was 15 g maltose (food-grade), 10 g glycerol, 10 g tryptone, 10 g yeast extract, 1 g KH 2 PO 4 , 0AE2 g MgSO 4 , and 0AE5 g CaCl 2 in 1 l of distilled water, while the order of effects of those components on exopolysaccharide production was yeast extract > maltose > tryptone > glycerol > KH 2 PO 4 > CaCl 2 > MgSO 4 , corresponding to the optimal concentration of medium was as follows: 20 g maltose (food-grade), 8 g glycerol, 5 g tryptone, 10 g yeast extract, 1 g KH 2 PO 4 , and 0AE5 g CaCl 2 in 1 l of distilled water. Conclusions: Under the optimal culture requirements, the maximum exopolysaccharide production reached 3AE5 g l )1 after 10 days of fermentation, while the maximum production of mycelial growth achieved 14AE5 g l )1 after 8 days of fermentation. Significance and Impact of the Study: This is the first report on the submerged culture requirements for mycelial growth and exopolysaccharide in C. jiangxiensis, and this two-step optimization strategy in this study can be widely applied to other microbial fermentation processes.

SignificancePhagocytosis and subsequent destruction of pathogens when the phagosomes in which they reside are fused with lysosomes are pillars of the eukaryotic innate immune defense. Consequently, evading trafficking to phagolysosomes is a fundamental survival strategy of most intracellular pathogens that replicate inside eukaryotic host cells. The obligatory intracellular bacterium Ehrlichia chaffeensis also avoids routing to host-cell phagolysosomes, but in a unique way: Ehrlichia secretes a protein, Ehrlichia translocated factor-2 (Etf-2), that has a Tre2-Bub2-Cdc16 (TBC)-like motif lacking RAB-GTPase–activating protein (GAP) activity. Etf-2 binds RAB5 on Ehrlichia inclusions and interferes with the engagement of RAB5-specific GAP with RAB5, thereby maintaining RAB5 in a GTP-bound active form on bacterial inclusions. Etf-2 is a unique example of a RAB-associated regulatory protein with a TBC-like motif lacking RABGAP activity.

AMT1-3 encodes the high affinity NH4+ transporter in rice roots and is predominantly expressed under nitrogen starvation. In order to evaluate the effect of AMT1-3 gene on rice growth, nitrogen absorption and metabolism, we generated AMT1-3-overexpressing plants and analyzed the growth phenotype, yield, carbon and nitrogen metabolic status, and gene expression profiles. Although AMT1-3 mRNA accumulated in transgenic plants, these plants displayed significant decreases in growth when compared to the wild-type plants. The nitrogen uptake assay using a 15N tracer revealed poor nitrogen uptake ability in AMT1-3-overexpressing plants. We found significant decreases in AMT1-3-overexpressing plant leaf carbon and nitrogen content accompanied with a higher leaf C/N ratio. Significant changes in soluble proteins and carbohydrates were also observed in AMT1-3-overexpressing plants. In addition, metabolite profile analysis demonstrated significant changes in individual sugars, organic acids and free amino acids. Gene expression analysis revealed distinct expression patterns of genes that participate in carbon and nitrogen metabolism. Additionally, the correlation between the metabolites and gene expression patterns was consistent in AMT1-3-overexpressing plants under both low and high nitrogen growth conditions. Therefore, we hypothesized that the carbon and nitrogen metabolic imbalance caused by AMT1-3 overexpressing attributed to the poor growth and yield of transgenic plants.

Polycephalomyces yunnanensis is described as a new species that parasitizes Ophiocordyceps nutans and stink bugs (hemipteran adults). It is characterized by the production of a viscous mass at the apex of its synnema, and it has two types of conidia (α- and β-conidia) and phialides (α- and β-phialides). A morphological and ecological investigation revealed that P. yunnanensis is distinct from any known species in the genus. Phylogenetic analyses based on the ITS and 5-locus (nrSSU, nrLSU, tef-1α, rpb1 and rpb2) data supported the treatment of this fungus as a distinct species.

A new species, Polycephalomyces agaricus, which is parasitic on Ophiocordyceps sp., a pathogen of melolonthid larvae in southwestern China, is described using morphological and phylogenetic evidence. This species differs morphologically from other species in the genus by its agaric-shaped synnemata, pileus-like slimy masses on short stipes, globose to subglobose α-conidia, and plenty of laterally or apically proliferating phialides. The phylogenetic analyses based on 5-locus (nrSSU, nrLSU, tef-1α, rpb1, and rpb2) data strongly support that this fungus is a distinct species in the genus.