Mechanisms of autophagosome biogenesis

Autophagy is a unique membrane trafficking process whereby newly formed membranes, termed phagophores, engulf parts of the cytoplasm leading to the production of double-membraned autophagosomes that get delivered to lysosomes for degradation. This catabolic pathway has been linked to numerous physiological and pathological conditions, such as development, programmed cell death, cancer, pathogen infection, neurodegenerative disorders, and myopathies. In this review, we will focus on recent studies in yeast and mammalian systems that have provided insights into two critical areas of autophagosome biogenesis - the source of the autophagosomal membranes, and the mechanisms regulating the fusion of the edges of the double-membraned phagophores to form autophagosomes.     

Foot-and-Mouth Disease Virus Induces Autophagosomes during Cell Entry via a Class III Phosphatidylinositol 3-Kinase-Independent Pathway

Autophagy is an intracellular pathway that can contribute to innate antiviral immunity by delivering viruses to lysosomes for degradation or can be beneficial for viruses by providing specialized membranes for virus replication. Here, we show that the picornavirus foot-and-mouth disease virus (FMDV) induces the formation of autophagosomes. Induction was dependent on Atg5, involved processing of LC3 to LC3II, and led to a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Furthermore, FMDV yields were reduced in cells lacking Atg5, suggesting that autophagy may facilitate FMDV infection. However, induction of autophagosomes by FMDV appeared to differ from starvation, as the generation of LC3 punctae was not inhibited by wortmannin, implying that FMDV-induced autophagosome formation does not require the class III phosphatidylinositol 3-kinase (PI3-kinase) activity of vps34. Unlike other picornaviruses, for which there is strong evidence that autophagosome formation is linked to expression of viral nonstructural proteins, FMDV induced autophagosomes very early during infection. Furthermore, autophagosomes could be triggered by either UV-inactivated virus or empty FMDV capsids, suggesting that autophagosome formation was activated during cell entry. Unlike other picornaviruses, FMDV-induced autophagosomes did not colocalize with the viral 3A or 3D protein. In contrast, ∼50% of the autophagosomes induced by FMDV colocalized with VP1. LC3 and VP1 also colocalized with the cellular adaptor protein p62, which normally targets ubiquitinated proteins to autophagosomes. These results suggest that FMDV induces autophagosomes during cell entry to facilitate infection, but not to provide membranes for replication.     

Pneumococcal capsular polysaccharide is immunogenic when present on the surface of macrophages and dendritic cells: TLR4 signaling induced by a conjugate vaccine or by lipopolysaccharide is conducive

Previously, we reported that a peptide, p458, from the sequence of the mammalian 60-kDa heat shock protein (hsp60) molecule can serve as a carrier in conjugate vaccines with capsular polysaccharide (CPS) molecules of various bacteria. These conjugate vaccines were effective injected in PBS without added adjuvants. We now report that p458 conjugated to pneumococcal CPS type 4 (PS4) manifests innate adjuvant effects: it stimulated mouse macrophages to secrete IL-12 and induced the late appearance of PS4 on the macrophage surface in a TLR4-dependent manner; PS4 alone or conjugated to other carriers did not stimulate macrophages in vitro. The injection of macrophages manifesting PS4 on the surface into mice induced long-term resistance to lethal Streptococcus pneumoniae challenge. The TLR4 ligand LPS could also induce the late appearance on the surface of unconjugated PS4 and resistance to challenge in injected mice. Resistance was not induced by macrophages containing only internalized PS4 or by pulsed macrophages that had been lysed. Glutaraldehyde-fixed macrophages pulsed with PS4 did induce resistance to lethal challenge. Moreover, bone marrow-derived dendritic cells activated by LPS and pulsed with unconjugated CPS were also effective in inducing resistance to lethal challenge. Resistance induced by the PS4-pulsed bone marrow-derived dendritic cell was specific for pneumococcal CPS serotypes (type 3 or type 4) and was associated with the induction of CPS-specific IgG and IgM Abs.     

A novel ROP/RAC GTPase effector integrates plant cell form and pattern formation

ROPs/RACs are the only known signaling Ras superfamily small GTPases in plants. As such they have been suggested to function as central regulators of diverse signaling cascades. The ROP/RAC signaling networks are largely unknown, however, because only few of their effector proteins have been identified. In a paper that was published in the June 5, 2007 issue of Current Biology we described the identification of a novel ROP/RAC effector designated ICR1 (Interactor of Constitutive active ROPs 1). We demonstrated that ICR1 functions as a scaffold that interacts with diverse but specific group of proteins including SEC3 subunit of the exocyst vesicle tethering complex. ICR1-SEC3 complexes can interact with ROPs in vivo and are thereby recruited to the plasma membrane. ICR1 knockdown or silencing leads to cell deformation and loss of the root stem cells population, and ectopic expression of ICR1 phenocopies activated ROPs/RACs. ICR1 presents a new paradigm in ROP/RAC signaling and integrates mechanisms regulating cell form and pattern formation at the whole plant level.Key words: Rho, auxin, root development, vesicle trafficking, RAC, ROP, polarity, Arabidopsis, exocyst     

Structural Organization and Energy Transduction Mechanism of Na+,K+-ATPase Studied with Transition Metal-Catalyzed Oxidative Cleavage

This chapter describes contributions of transition metal-catalyzed oxidative cleavage of Na⁺,K⁺-ATPase to our understanding of structure–function relations. In the presence of ascorbate/H₂O₂, specific cleavages are catalyzed by the bound metal and because more than one peptide bond close to the metal can be cleaved, this technique reveals proximity of the different cleavage positions within the native structure. Specific cleavages are catalyzed by Fe²⁺ bound at the cytoplasmic surface or by complexes of ATP–Fe²⁺, which directs the Fe²⁺ to the normal ATP–Mg²⁺ site. Fe²⁺- and ATP–Fe²⁺-catalyzed cleavages reveal large conformation-dependent changes in interactions between cytoplasmic domains, involving conserved cytoplasmic sequences, and a change of ligation of Mg²⁺ ions between E₁P and E₂P, which may be crucial in facilitating hydrolysis of E₂P. The pattern of domain interactions in E₁ and E₂ conformations, and role of Mg²⁺ ions, may be common to all P-type pumps. Specific cleavages can also be catalyzed by Cu²⁺ ions, bound at the extracellular surfaces, or a hydrophobic Cu²⁺-diphenyl phenanthroline (DPP) complex, which directs the Cu²⁺ to the membrane–water interface. Cu²⁺- or Cu²⁺-DPP-catalyzed cleavages are providing information on / subunit interactions and spatial organization of transmembrane segments. Transition metal-catalyzed cleavage could be widely used to investigate other P-type pumps and membrane proteins and, especially, ATP binding proteins.     

Intra-Golgi protein transport depends on a cholesterol balance in the lipid membrane

Transport of proteins between intracellular membrane compartments is mediated by a protein machinery that regulates the budding and fusion processes of individual transport steps. Although the core proteins of both processes are defined at great detail, much less is known about the involvement of lipids. Here we report that changing the cellular balance of cholesterol resulted in changes of the morphology of the Golgi apparatus, accompanied by an inhibition of protein transport. By using a well characterized cell-free intra-Golgi transport assay, these observations were further investigated, and it was found that the transport reaction is sensitive to small changes in the cholesterol content of Golgi membranes. Addition as well as removal of cholesterol (10 +/- 6%) to Golgi membranes by use of methyl-beta-cyclodextrin specifically inhibited the intra-Golgi transport assay. Transport inhibition occurred at the fusion step. Modulation of the cholesterol content changed the lipid raft partitioning of phosphatidylcholine and heterotrimeric G proteins, but not of other (non) lipid raft proteins and lipids. We suggest that the cholesterol balance in Golgi membranes plays an essential role in intra-Golgi protein transport and needs to be carefully regulated to maintain the structural and functional organization of the Golgi apparatus.     

Early expression of interferon gamma following oral antigen administration is associated with peripheral tolerance induction

Oral tolerance is the induction of immune hyporesponsiveness to orally administered antigens. It was described in association with a decrease in interferon gamma (IFNgamma) production by activated T cells. To determine the role of IFNgamma and IL10 in immunemodulation via oral tolerization. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Treated mice received five oral doses of colitis-extracted proteins (CEPs) every other day, starting immediately after colitis induction. Control mice received similar doses of bovine serum albumin. Colitis was assessed in both groups by standard clinical, micro- and macroscopic scores. IFNgamma and IL10 expression in splenic lymphocytes from both groups was tested by RT-PCR immediately after oral feeding, 1, 4, 8, 12, and 24 h thereafter and then every 24 h for 2 weeks. Feeding of CEPs markedly ameliorated experimental colitis. These mice gained weight and showed markedly improved macro- and microscopic parameters of colitis. Tolerized mice exhibited IFNgamma expression in splenic lymphocytes starting immediately following oral CEP immunization and up to 14 d thereafter. IL10 was expressed starting 1 h after CEP feeding and during the first 48 h thereafter. In contrast, non-tolerized control mice manifested IFNgamma expression starting on day 6 and had no IL10 expression. Early induction of IFNgamma expression by oral antigen may be associated with systemic tolerance in the experimental colitis setting. In contrast, late expression of IFNgamma is associated with a pro-inflammatory response in non-tolerized controls.     

Aut7p, a soluble autophagic factor, participates in multiple membrane trafficking processes

Aut7p, a protein recently implicated in autophagic events in the yeast Saccharomyces cerevisiae, exhibits significant homology to a mammalian protein, p16, herein termed GATE-16 (Golgi-associated ATPase Enhancer of 16 kDa), a novel intra-Golgi transport factor. Here we provide evidence for the involvement of Aut7p in different membrane trafficking processes. Aut7p largely substitutes for the activity of GATE-16 in mammalian intra-Golgi transport in vitro. In vivo, AUT7 interacts genetically with endoplasmic reticulum to Golgi SNAREs, specifically with BET1 and SEC22. Aut7p interacts physically with the following two v-SNAREs: Bet1p, which is involved in endoplasmic reticulum to Golgi vesicular transport, and Nyv1p, implicated in vacuolar inheritance. We suggest that, in addition to its role in autophagocytosis, Aut7p has pleiotropic effects and participates in at least two membrane traffic events.     

Intracellular retention and degradation of the epidermal growth factor receptor, two distinct processes mediated by benzoquinone ansamycins

Epidermal growth factor (EGF) stimulates the growth of various types of cells via its cell surface tyrosine kinase receptor. The EGF receptor (EGF-R) has an oncogenic potential when overexpressed in a wide range of tumor cells. Geldanamycin (GA) and herbimycin (HA), specific inhibitors of the cytosolic chaperone HSP 90 and its endoplasmic reticulum homologue GRP 94, were shown to accelerate degradation of the EGF-R and of its homologue p185(c-)(erbB-2). Here we compared the effects of GA and HA on intracellular degradation and maturation of EGF-R. By using an inhibitor of proteasomal degradation, we learned that GA, but not HA, blocks processing of newly synthesized EGF-R. The effects of GA and HA on receptor degradation are mediated by the cytosolic portion of EGF-R and could be conferred to the erythropoietin receptor (EPO-R), by employing the respective chimera. Neither HA nor GA affected stability of newly synthesized EGF-R lacking the cytosolic domain (Ex EGF-R), but GA caused intracellular retention of this mutant. Taken together, our results imply that GA has two distinct targets of action on the EGF-R, one for promoting its degradation and another for mediating its intracellular retention. Apparently, degradation of the EGF-R mediated by GA or HA requires the presence of the EGF-R cytosolic domain, whereas intracellular retention in the presence of GA is coupled to the extracellular domain of the EGF-R.     

Structure of GATE-16, membrane transport modulator and mammalian ortholog of autophagocytosis factor Aut7p

The GATE-16 protein participates in intra-Golgi transport and can associate with the N-ethylmaleimide-sensitive fusion protein and with Golgi SNAREs. The yeast ortholog of GATE-16 is the autophagocytosis factor Aut7p. GATE-16 is also closely related to the GABA receptor-associated protein (GABARAP), which has been proposed to cluster neurotransmitter receptors by mediating interaction with the cytoskeleton, and to the light chain-3 subunit of the neuronal microtubule-associated protein complex. Here, we present the crystal structure of GATE-16 refined to 1.8 A resolution. GATE-16 contains a ubiquitin fold decorated by two additional N-terminal helices. Proteins with strong structural similarity but no detectable sequence homology to GATE-16 include Ras effectors that mediate diverse downstream functions, but each interacts with Ras by forming pseudo-continuous beta-sheets. The GATE-16 surface suggests that it binds its targets in a similar manner. Moreover, a second potential protein-protein interaction site on GATE-16 may explain the adapter activity observed for members of the GATE-16 family.