FREE1 regulates phagophore closure in plants.

Adenosine monophosphate-activated protein kinase (AMPK), the central energy sensor in more complex eukaryotes, can activate macroautophagy/autophagy upon cellular energy deficiency. However, the regulatory role of nutrient sensing in mediating phagophore closure to generate an autophagosome remains unknown. The evolutionarily conserved endosomal sorting complexes required for transport (ESCRT) machinery has been postulated to regulate phagophore sealing, yet the signaling pathway modulating the ESCRT complex relocation from multivesicular body (MVB) to phagophore for closure remains unknown. We recently identified a plant unique pleiotropic protein FREE1 (FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING 1), which is phosphorylated by the plant energy sensor SnRK1 (SNF1-related kinase 1) and bridges the ATG conjugation system and ESCRT machinery to regulate phagophore sealing upon nutrient starvation. This study elucidated the bona fide roles and underlying mechanism of cellular energy-sensing pathways in regulating compartment sealing.

A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress.

Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.

For cells to survive they need to be able to remove faulty or damaged components. The ability to recycle faulty parts is so crucial that some of the molecular machinery responsible is the same across the plant and animal kingdoms. One of the major recycling pathways cells use is autophagy, which labels damaged proteins with molecular tags that say 'eat-me'. Proteins called receptors then recognize these tags and move the faulty component into vesicles that transport the cargo to a specialized compartment that recycles broken parts. Cells make and fold around 40% of their proteins at a site called the endoplasmic reticulum, or ER for short. However, the process of folding and synthesizing proteins is prone to errors. For example, when a cell is under stress this can cause a 'stall' in production, creating a build-up of faulty, partially constructed proteins that are toxic to the cell. There are several quality control systems which help recognize and correct these errors in production. Yet, it remained unclear how autophagy and these quality control mechanisms are linked together. Here, Stephani, Picchianti et al. screened for receptors that regulate the recycling of faulty proteins by binding to the 'eat-me' tags. This led to the identification of a protein called C53, which is found in both plant and animal cells. Microscopy and protein-protein interaction tests showed that C53 moves into transport vesicles when the ER is under stress and faulty proteins start to build-up. Once there, C53 interacts with two proteins embedded in the wall of the endoplasmic reticulum. These proteins form part of the quality control system that senses stalled protein production, labelling the stuck proteins with 'eat-me' tags. Together with C53, they identify and remove half-finished proteins before they can harm the cell. The fact that C53 works in the same way in both plant and human cells suggests that many species might use this receptor to recycle stalled proteins. This has implications for a wide range of research areas, from agriculture to human health. A better understanding of C53 could be beneficial for developing stress-resilient crops. It could also aid research into human diseases, such as cancer and viral infections, that have been linked to C53 and its associated proteins.

ClpP-deletion impairs the virulence of Legionella pneumophila and the optimal translocation of effector proteins.

BACKGROUND: The opportunistic bacterial pathogen Legionella pneumophila uses substrate effectors of Dot/Icm type IVB secretion system (T4BSS) to accomplish survival and replication in amoebae cells and mammalian alveolar macrophages. During the conversion between its highly resistant, infectious dormant form and vigorously growing, uninfectious replicative form, L. pneumophila utilizes a complicated regulatory network in which proteolysis may play a significant role. As a highly conserved core protease, ClpP is involved in various cellular processes as well as virulence in bacteria, and has been proved to be required for the expression of transmission traits and cell division of L. pneumophila. RESULTS: The clpP-deficient L. pneumophila strain failed to replicate and was digested in the first 3 h post-infection in mammalian cells J774A.1. Further investigation demonstrates that the clpP deficient mutant strain was unable to escape the endosome-lysosomal pathway in host cells. We also found that the clpP deficient mutant strain still expresses T4BSS components, induces contact-dependent cytotoxicity and translocate effector proteins RalF and LegK2, indicating that its T4BSS was overall functional. Interestingly, we further found that the translocation of several effector proteins is significantly reduced without ClpP. CONCLUSIONS: The data indicate that ClpP plays an important role in regulating the virulence and effector translocation of Legionella pneumophila.

How vacuolar sorting receptor proteins interact with their cargo proteins: crystal structures of apo and cargo-bound forms of the protease-associated domain from an Arabidopsis vacuolar sorting receptor.

In plant cells, soluble proteins are directed to vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptors (VSR). To understand how a VSR recognizes its cargo, we present the crystal structures of the protease-associated domain of VSR isoform 1 from Arabidopsis thaliana (VSR1PA) alone and complexed with a cognate peptide containing the barley (Hordeum vulgare) aleurain VSD sequence of 1ADSNPIRPVT10. The crystal structures show that VSR1PA binds the sequence, Ala-Asp-Ser, preceding the NPIR motif. A conserved cargo binding loop, with a consensus sequence of 95RGxCxF100, forms a cradle that accommodates the cargo-peptide. In particular, Arg-95 forms a hydrogen bond to the Ser-3 position of the VSD, and the essential role of Arg-95 and Ser-3 in receptor-cargo interaction was supported by a mutagenesis study. Cargo binding induces conformational changes that are propagated from the cargo binding loop to the C terminus via conserved residues in switch I-IV regions. The resulting 180° swivel motion of the C-terminal tail is stabilized by a hydrogen bond between Glu-24 and His-181. A mutagenesis study showed that these two residues are essential for cargo interaction and trafficking. Based on our structural and functional studies, we present a model of how VSRs recognize their cargos.

Trans-Golgi network-located AP1 gamma adaptins mediate dileucine motif-directed vacuolar targeting in Arabidopsis.

Membrane proteins on the tonoplast are indispensible for vacuolar functions in plants. However, how these proteins are transported to the vacuole and how they become separated from plasma membrane proteins remain largely unknown. In this study, we used Arabidopsis thaliana vacuolar ion transporter1 (VIT1) as a reporter to study the mechanisms of tonoplast targeting. We showed that VIT1 reached the tonoplast through a pathway involving the endoplasmic reticulum (ER), Golgi, trans-Golgi network (TGN), prevacuolar compartment, and tonoplast. VIT1 contains a putative N-terminal dihydrophobic type ER export signal, and its N terminus has a conserved dileucine motif (EKQTLL), which is responsible for tonoplast targeting. In vitro peptide binding assays with synthetic VIT1 N terminus identified adaptor protein complex-1 (AP1) subunits that interacted with the dileucine motif. A deficiency of AP1 gamma adaptins in Arabidopsis cells caused relocation of tonoplast proteins containing the dileucine motif, such as VIT1 and inositol transporter1, to the plasma membrane. The dileucine motif also effectively rerouted the plasma membrane protein SCAMP1 to the tonoplast. Together with subcellular localization studies showing that AP1 gamma adaptins localize to the TGN, we propose that the AP1 complex on the TGN mediates tonoplast targeting of membrane proteins with the dileucine motif.

The Legionella pneumophila Dps homolog is regulated by iron and involved in multiple stress tolerance.

Iron homeostasis is essential to almost all organisms. In this study, we identified the putative homolog of the iron-storage protein-encoding gene, dpsL, in the intracellular pathogen Legionella pneumophila and demonstrated its expression under iron-limited conditions and its responses to multiple stresses. Quantitative real-time PCR analysis indicated that the expression of dpsL was enhanced under iron limitation regardless of the growth phase. Compared with the wild-type cells, the cells devoid of dpsL were heat and H(2)O(2)-sensitive. In contrast to the dps mutants of other bacteria, the growth of the dpsL mutant in an iron-deprived medium was delayed but finally reached the same cell density as wild-type cells during the stationary phase of growth. The finding that the dpsL mutant is salt resistant suggested the involvement of DpsL in virulence.